Development of an α-synuclein positron emission tomography tracer for imaging synucleinopathies.

Synucleinopathies are characterized by the accumulation of α-synuclein (α-Syn) aggregates in the brain. Positron emission tomography (PET) imaging of synucleinopathies requires radiopharmaceuticals that selectively bind α-Syn deposits. We report the identification of a brain permeable and rapid washout PET tracer [18F]-F0502B, which shows high binding affinity for α-Syn, but not for Aß or Tau fibrils, and preferential binding to α-Syn aggregates in the brain sections. Employing several cycles of counter screenings with in vitro fibrils, intraneuronal aggregates, and neurodegenerative disease brain sections from several mice models and human subjects, [18F]-F0502B images α-Syn deposits in the brains of mouse and non-human primate PD models. We further determined the atomic structure of the α-Syn fibril-F0502B complex by cryo-EM and revealed parallel diagonal stacking of F0502B on the fibril surface through an intense noncovalent bonding network via inter-ligand interactions. Therefore, [18F]-F0502B is a promising lead compound for imaging aggregated α-Syn in synucleinopathies.

Regulation and functions of non-m6A mRNA modifications.

Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N6-methyladenosine (m6A), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-m6A modifications in eukaryotic mRNA, including pseudouridine (Ψ), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), inosine, 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2'-O-methylated nucleotide (Nm) and internal N7-methylguanosine (m7G). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-m6A mRNA modifications and discuss their potential future applications.

Phase Separation of Disease-Associated SHP2 Mutants Underlies MAPK Hyperactivation.

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.

Better Together: A Hybrid Amyloid Signals Necroptosis.

A new solid-state NMR study determines the high-resolution hetero-amyloid structure of the RIPK1-RIPK3 signaling complex that is involved in mediating necroptosis. The structure demonstrates specific formation of hetero-amyloids over homo-amyloids and the structural basis for a functional amyloid to act as a platform to recruit and activate downstream partners in intracellular signaling.

PARP5A and RNF146 phase separation restrains RIPK1-dependent necroptosis.

Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.

Stress Induces Dynamic, Cytotoxicity-Antagonizing TDP-43 Nuclear Bodies via Paraspeckle LncRNA NEAT1-Mediated Liquid-Liquid Phase Separation.

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.

Conformational strains of pathogenic amyloid proteins in neurodegenerative diseases.

Amyloid proteins, which are considered 'villains' in many neurodegenerative diseases, form enigmatic pathological strains that underlie disease pathogenesis and progression. Recent technical advances in cryogenic electron microscopy and solid-state NMR spectroscopy have enabled the high-resolution structures of full-length amyloid fibrils to be determined, initiating an era in which we have the opportunity to gain atomic-level structural understanding of pathogenic protein aggregation in neurodegenerative diseases. In this Review, we aim to explain the clinicopathological heterogeneity of neurodegenerative diseases by considering the polymorphic structures of amyloid fibrils. We decipher the structural basis for the generation of fibril polymorphs, how the fibril polymorphs differ in different disease contexts and how conformational changes alter the pathology caused by amyloid proteins during disease progression. Finally, we evaluate how this knowledge might aid clinical diagnostic and therapeutic strategies to treat neurodegenerative diseases.

The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

Prospective, multi-site study of healthcare utilization after actionable monogenic findings from clinical sequencing.

As large-scale genomic screening becomes increasingly prevalent, understanding the influence of actionable results on healthcare utilization is key to estimating the potential long-term clinical impact. The eMERGE network sequenced individuals for actionable genes in multiple genetic conditions and returned results to individuals, providers, and the electronic health record. Differences in recommended health services (laboratory, imaging, and procedural testing) delivered within 12 months of return were compared among individuals with pathogenic or likely pathogenic (P/LP) findings to matched individuals with negative findings before and after return of results. Of 16,218 adults, 477 unselected individuals were found to have a monogenic risk for arrhythmia (n = 95), breast cancer (n = 96), cardiomyopathy (n = 95), colorectal cancer (n = 105), or familial hypercholesterolemia (n = 86). Individuals with P/LP results more frequently received services after return (43.8%) compared to before return (25.6%) of results and compared to individuals with negative findings (24.9%; p < 0.0001). The annual cost of qualifying healthcare services increased from an average of $162 before return to $343 after return of results among the P/LP group (p < 0.0001); differences in the negative group were non-significant. The mean difference-in-differences was $149 (p < 0.0001), which describes the increased cost within the P/LP group corrected for cost changes in the negative group. When stratified by individual conditions, significant cost differences were observed for arrhythmia, breast cancer, and cardiomyopathy. In conclusion, less than half of individuals received billed health services after monogenic return, which modestly increased healthcare costs for payors in the year following return.

The NS2B-PP1α-eIF2α axis: Inhibiting stress granule formation and Boosting Zika virus replication.

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

Phase-separated ParB enforces diverse DNA compaction modes and stabilizes the parS-centered partition complex.

The tripartite ParABS system mediates chromosome segregation in the majority of bacterial species. Typically, DNA-bound ParB proteins around the parS sites condense the chromosomal DNA into a higher-order multimeric nucleoprotein complex for the ParA-driven partition. Despite extensive studies, the molecular mechanism underlying the dynamic assembly of the partition complex remains unclear. Herein, we demonstrate that Bacillus subtilis ParB (Spo0J), through the multimerization of its N-terminal domain, forms phase-separated condensates along a single DNA molecule, leading to the concurrent organization of DNA into a compact structure. Specifically, in addition to the co-condensation of ParB dimers with DNA, the engagement of well-established ParB condensates with DNA allows for the compression of adjacent DNA and the looping of distant DNA. Notably, the presence of CTP promotes the formation of condensates by a low amount of ParB at parS sites, triggering two-step DNA condensation. Remarkably, parS-centered ParB-DNA co-condensate constitutes a robust nucleoprotein architecture capable of withstanding disruptive forces of tens of piconewton. Overall, our findings unveil diverse modes of DNA compaction enabled by phase-separated ParB and offer new insights into the dynamic assembly and maintenance of the bacterial partition complex.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.

BACKGROUND: Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1-interacting protein of 55 kDa) protects against HF. METHODS: Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid-liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on ß-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 in HF. RESULTS: Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the ß-adrenergic receptor-mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF. CONCLUSIONS: Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system-mediated heart failure.

OARD: Open annotations for rare diseases and their phenotypes based on real-world data.

Diagnosis for rare genetic diseases often relies on phenotype-driven methods, which hinge on the accuracy and completeness of the rare disease phenotypes in the underlying annotation knowledgebase. Existing knowledgebases are often manually curated with additional annotations found in published case reports. Despite their potential, real-world data such as electronic health records (EHRs) have not been fully exploited to derive rare disease annotations. Here, we present open annotation for rare diseases (OARD), a real-world-data-derived resource with annotation for rare-disease-related phenotypes. This resource is derived from the EHRs of two academic health institutions containing more than 10 million individuals spanning wide age ranges and different disease subgroups. By leveraging ontology mapping and advanced natural-language-processing (NLP) methods, OARD automatically and efficiently extracts concepts for both rare diseases and their phenotypic traits from billing codes and lab tests as well as over 100 million clinical narratives. The rare disease prevalence derived by OARD is highly correlated with those annotated in the original rare disease knowledgebase. By performing association analysis, we identified more than 1 million novel disease-phenotype association pairs that were previously missed by human annotation, and >60% were confirmed true associations via manual review of a list of sampled pairs. Compared to the manual curated annotation, OARD is 100% data driven and its pipeline can be shared across different institutions. By supporting privacy-preserving sharing of aggregated summary statistics, such as term frequencies and disease-phenotype associations, it fills an important gap to facilitate data-driven research in the rare disease community.

Stripe rust effector Pst03724 modulates host immunity by inhibiting NAD kinase activation by a calmodulin.

Puccinia striiformis f. sp. tritici (Pst) secretes effector proteins that enter plant cells to manipulate host immune processes. In this report, we present an important Pst effector, Pst03724, whose mRNA expression level increases during Pst infection of wheat (Triticum aestivum). Silencing of Pst03724 reduced the growth and development of Pst. Pst03724 targeted the wheat calmodulin TaCaM3-2B, a positive regulator of wheat immunity. Subsequent investigations revealed that Pst03724 interferes with the TaCaM3-2B-NAD kinase (NADK) TaNADK2 association and thus inhibits the enzyme activity of TaNADK2 activated by TaCaM3-2B. Knocking down TaNADK2 expression by virus-mediated gene silencing significantly increased fungal growth and development, suggesting a decrease in resistance against Pst infection. In conclusion, our findings indicate that Pst effector Pst03724 inhibits the activity of NADK by interfering with the TaCaM3-2B-TaNADK2 association, thereby facilitating Pst infection.

Structural mechanism for specific binding of chemical compounds to amyloid fibrils.

Amyloid fibril is an important pharmaceutical target for diagnostic and therapeutic treatment of neurodegenerative diseases. However, rational design of chemical compounds that interact with amyloid fibrils is unachievable due to the lack of mechanistic understanding of the ligand-fibril interaction. Here we used cryoelectron microscopy to survey the amyloid fibril-binding mechanism of a series of compounds including classic dyes, (pre)clinical imaging tracers and newly identified binders from high-throughput screening. We obtained clear densities of several compounds in complex with an α-synuclein fibril. These structures unveil the basic mechanism of the ligand-fibril interaction, which exhibits remarkable difference from the canonical ligand-protein interaction. In addition, we discovered a druggable pocket that is also conserved in the ex vivo α-synuclein fibrils from multiple system atrophy. Collectively, these findings expand our knowledge of protein-ligand interaction in the amyloid fibril state, which will enable rational design of amyloid binders in a medicinally beneficial way.

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments.

Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies and the creation of harmful intermediates. Detailed visualization of this misfolding process is of paramount importance for improving our understanding of disease mechanisms and for the development of potential therapeutic strategies. While in vitro studies using purified proteins have been instrumental in delivering significant insights into protein misfolding, the behavior of these proteins in the complex milieu of living cells often diverges significantly from such simplified environments. Biomedical imaging performed in cell provides cellular-level information with high physiological and pathological relevance, often surpassing the depth of information attainable through in vitro methods. This review highlights a variety of methodologies used to scrutinize protein misfolding within biological systems. This includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, and cryo-electron microscopy. Recent advancements in these techniques have notably deepened our understanding of protein misfolding processes and the features of the resulting misfolded species within living cells. The progression in these fields promises to catalyze further breakthroughs in our comprehension of neurodegenerative disease mechanisms and potential therapeutic interventions.

Symmetric control of sister chromatid cohesion establishment.

Besides entrapping sister chromatids, cohesin drives other high-order chromosomal structural dynamics like looping, compartmentalization and condensation. ESCO2 acetylates a subset of cohesin so that cohesion must be established and only be established between nascent sister chromatids. How this process is precisely achieved remains unknown. Here, we report that GSK3 family kinases provide higher hierarchical control through an ESCO2 regulator, CRL4MMS22L. GSK3s phosphorylate Thr105 in MMS22L, resulting in homo-dimerization of CRL4MMS22L and ESCO2 during S phase as evidenced by single-molecule spectroscopy and several biochemical approaches. A single phospho-mimicking mutation on MMS22L (T105D) is sufficient to mediate their dimerization and rescue the cohesion defects caused by GSK3 or MMS22L depletion, whereas non-phosphorylable T105A exerts dominant-negative effects even in wildtype cells. Through cell fractionation and time-course measurements, we show that GSK3s facilitate the timely chromatin association of MMS22L and ESCO2 and subsequently SMC3 acetylation. The necessity of ESCO2 dimerization implicates symmetric control of cohesion establishment in eukaryotes.

Global profiling of arginine dimethylation in regulating protein phase separation by a steric effect-based chemical-enrichment method.

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid-liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect-based chemical-enrichment method (SECEM) coupled with liquid chromatography-tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG-rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG-rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.