Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography-high-resolution tandem mass spectrometry.

Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.

Exploring the effective components of honey-processed licorice (Glycyrrhiza uralensis Fisch.) in attenuating Doxorubicin-induced myocardial cytotoxicity by combining network pharmacology and in vitro experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is widely used clinically as one of the most famous traditional Chinese herbs. Its herb roasted with honey is called honey-processed licorice (HPL). Modern studies have shown that HPL has a stronger cardioprotective ability compared to raw licorice (RL), however the material basis and mechanism of action of the potential cardioprotection have not been fully elucidated. AIM OF THE STUDY: To screen and validate the material basis of cardioprotection exerted by HPL and to preliminarily predict the potential mechanism of action. MATERIALS AND METHODS: UPLC-QTOF-MS/MS was used to analyze HPL samples with different processing levels, and differential compounds were screened out through principal component analysis. Network pharmacology and molecular docking were applied to explore the association between differential compounds and doxorubicin cardiomyopathy and their mechanisms of action were predicted. An in vitro model was established to verify the cardioprotective effects of differential compounds. RESULTS: Six differential compounds were screened as key components of HPL for potential cardioprotection. Based on network pharmacology, 113 potential important targets for the treatment of Dox-induced cardiotoxicity were screened. KEGG enrichment analysis predicted that the PI3K-Akt pathway was closely related to the mechanism of action of active ingredients. Molecular docking results showed that the six differential compounds all had good binding activity with Nrf2 protein. In addition, in vitro experiments had shown that five of the active ingredients (liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and licochalcone A) can significantly increase Dox-induced H9c2 cell viability, SOD activity, and mitochondrial membrane potential, significantly reduces MDA levels and inhibits ROS generation. CONCLUSION: Liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin and licochalcone A are key components of HPL with potential cardioprotective capabilities. Five active ingredients can alleviate Dox-induced cardiotoxicity by inhibiting oxidative stress and mitochondrial damage.

HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

Prediction of Clinical Precision Chemotherapy by Patient-Derived 3D Bioprinting Models of Colorectal Cancer and Its Liver Metastases.

Methods accurately predicting the responses of colorectal cancer (CRC) and colorectal cancer liver metastasis (CRLM) to personalized chemotherapy remain limited due to tumor heterogeneity. This study introduces an innovative patient-derived CRC and CRLM tumor model for preclinical investigation, utilizing 3d-bioprinting (3DP) technology. Efficient construction of homogeneous in vitro 3D models of CRC/CRLM is achieved through the application of patient-derived primary tumor cells and 3D bioprinting with bioink. Genomic and histological analyses affirm that the CRC/CRLM 3DP tumor models effectively retain parental tumor biomarkers and mutation profiles. In vitro tests evaluating chemotherapeutic drug sensitivities reveal substantial tumor heterogeneity in chemotherapy responses within the 3DP CRC/CRLM models. Furthermore, a robust correlation is evident between the drug response in the CRLM 3DP model and the clinical outcomes of neoadjuvant chemotherapy. These findings imply a significant potential for the application of patient-derived 3DP cancer models in precision chemotherapy prediction and preclinical research for CRC/CRLM.

Chromosome-scale genomes of five Hongmu species in Leguminosae.

The Legume family (Leguminosae or Fabaceae), is one of the largest and economically important flowering plants. Heartwood, the core of a tree trunk or branch, is a valuable and renewable resource employed for centuries in constructing sturdy and sustainable structures. Hongmu refers to a category of precious timber trees in China, encompassing 29 woody species, primarily from the legume genus. Due to the lack of genome data, detailed studies on their economic and ecological importance are limited. Therefore, this study generates chromosome-scale assemblies of five Hongmu species in Leguminosae: Pterocarpus santalinus, Pterocarpus macrocarpus, Dalbergia cochinchinensis, Dalbergia cultrata, and Senna siamea, using a combination of short-reads, long-read nanopore, and Hi-C data. We obtained 623.86 Mb, 634.58 Mb, 700.60 Mb, 645.98 Mb, and 437.29 Mb of pseudochromosome level assemblies with the scaffold N50 lengths of 63.1 Mb, 63.7 Mb, 70.4 Mb, 61.1 Mb and 32.2 Mb for P. santalinus, P. macrocarpus, D. cochinchinensis, D. cultrata and S. siamea, respectively. These genome data will serve as a valuable resource for studying crucial traits, like wood quality, disease resistance, and environmental adaptation in Hongmu.

Neurodevelopmental toxicity of bisphenol AF in zebrafish larvae and the protective effects of curcumin.

Bisphenol AF (BPAF) is one of the most commonly used alternatives of bisphenol A in the plastics industry. The effects of BPAF on nervous development are unclear. Curcumin (CUR) has been determined to be an anti-inflammatory and antioxidant agent. In this study, the effects of BPAF on neurotoxicity of zebrafish embryos/larvae and whether CUR could reverse effects induced by BPAF were investigated. The results showed that BPAF treatment induced deficits in locomotor behavior, altered the larval brain development, caused aberrant expression of neurogenesis related genes (elavl3, zn5, α-tubulin, syn2a, and gap43), decreased acetylcholinesterase (AChE) activity, and induced oxidative stress, cell apoptosis, and neuroinflammation in zebrafish larvae. CUR addition could block the adverse effects of BPAF on nervous development by attenuated oxidative stress and cell apoptosis induced by BPAF in zebrafish, enhanced the activity of AChE, and increased the expression of genes involved in the pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α, and IL-8). The results of this study indicate that BPAF could induce aberrant development on nervous system. However, CUR exerts neuroprotective effects on BPAF-induced neurotoxicity in zebrafish larvae.

Case report: A heterozygous mutation in ZNF462 leads to growth hormone deficiency.

Weiss-Kruszka syndrome (WSKA) is a rare disease most often caused by mutations in the ZNF462 gene. To screen for hereditary diseases, exons from the patient's genome were sequenced. Genomic PCR experiments followed by Sanger sequencing were used to confirm the mutated genomic regions in the patient and his parents. We report a new mutation site, a heterozygous mutation (NM_021224.6:c.6311dup) in ZNF462 in a male patient of 8 years old. The mutation in the ZNF462 gene caused WSKA. This patient is the first case with WSKA characterized by attention-deficit hyperactivity disorder and complete growth hormone deficiency without pituitary lesions. Our results suggest that the heterozygous mutation in ZNF462 is the direct cause of WSKA in this patient. Mutations in other genes interacting with ZNF462 result in similar symptoms of WSKA. Furthermore, ZNF462 and its interacting proteins ASXL2 and VPS13B may form a protein complex that is important for normal development but awaits more studies to reveal its detailed functions.

Quantum Circuit Optimization for Solving Discrete Logarithm of Binary Elliptic Curves Obeying the Nearest-Neighbor Constrained.

In this paper, we consider the optimization of the quantum circuit for discrete logarithm of binary elliptic curves under a constrained connectivity, focusing on the resource expenditure and the optimal design for quantum operations such as the addition, binary shift, multiplication, squaring, inversion, and division included in the point addition on binary elliptic curves. Based on the space-efficient quantum Karatsuba multiplication, the number of CNOTs in the circuits of inversion and division has been reduced with the help of the Steiner tree problem reduction. The optimized size of the CNOTs is related to the minimum degree of the connected graph.

RING finger 138 deregulation distorts NF-кB signaling and facilities colitis switch to aggressive malignancy.

Prolonged activation of nuclear factor (NF)-кB signaling significantly contributes to the development of colorectal cancer (CRC). New therapeutic opportunities are emerging from targeting this distorted cell signaling transduction. Here, we discovered the critical role of RING finger 138 (RNF138) in CRC tumorigenesis through regulating the NF-кB signaling, which is independent of its Ubiquitin-E3 ligase activity involved in DNA damage response. RNF138-/- mice were hyper-susceptible to the switch from colitis to aggressive malignancy, which coincided with sustained aberrant NF-кB signaling in the colonic cells. Furthermore, RNF138 suppresses the activation of NF-кB signaling pathway through preventing the translocation of NIK and IKK-Beta Binding Protein (NIBP) to the cytoplasm, which requires the ubiquitin interaction motif (UIM) domain. More importantly, we uncovered a significant correlation between poor prognosis and the downregulation of RNF138 associated with reinforced NF-кB signaling in clinical settings, raising the possibility of RNF138 dysregulation as an indicator for the therapeutic intervention targeting NF-кB signaling. Using the xenograft models built upon either RNF138-dificient CRC cells or the cells derived from the RNF138-dysregulated CRC patients, we demonstrated that the inhibition of NF-кB signaling effectively hampered tumor growth. Overall, our work defined the pathogenic role of aberrant NF-кB signaling due to RNF138 downregulation in the cascade events from the colitis switch to colonic neoplastic transformation and progression, and also highlights the possibility of targeting the NF-кB signaling in treating specific subtypes of CRC indicated by RNF138-ablation.

The Deficits of Individual Morphological Covariance Network Architecture in Schizophrenia Patients With and Without Violence.

Background: Schizophrenia is associated with a significant increase in the risk of violence, which constitutes a public health concern and contributes to stigma associated with mental illness. Although previous studies revealed structural and functional abnormalities in individuals with violent schizophrenia (VSZ), the neural basis of psychotic violence remains controversial. Methods: In this study, high-resolution structural magnetic resonance imaging (MRI) data were acquired from 18 individuals with VSZ, 23 individuals with non-VSZ (NSZ), and 22 age- and sex-matched healthy controls (HCs). Whole-brain voxel-based morphology and individual morphological covariance networks were analysed to reveal differences in gray matter volume (GMV) and individual morphological covariance network topology. Relationships among abnormal GMV, network topology, and clinical assessments were examined using correlation analyses. Results: GMV in the hypothalamus gradually decreased from HCs and NSZ to VSZ and showed significant differences between all pairs of groups. Graph theory analyses revealed that morphological covariance networks of HCs, NSZ, and VSZ exhibited small worldness. Significant differences in network topology measures, including global efficiency, shortest path length, and nodal degree, were found. Furthermore, changes in GMV and network topology were closely related to clinical performance in the NSZ and VSZ groups. Conclusions: These findings revealed the important role of local structural abnormalities of the hypothalamus and global network topological impairments in the neuropathology of NSZ and VSZ, providing new insight into the neural basis of and markers for VSZ and NSZ to facilitate future accurate clinical diagnosis and targeted treatment.

Correction to: Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

An amendment to this paper has been published and can be accessed via the original article.

PDCD6 cooperates with C-Raf to facilitate colorectal cancer progression via Raf/MEK/ERK activation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies, and it's expected that the CRC burden will substantially increase in the next two decades. New biomarkers for targeted treatment and associated molecular mechanism of tumorigenesis remain to be explored. In this study, we investigated whether PDCD6 plays an oncogenic role in colorectal cancer and its underlying mechanism. METHODS: Programmed cell death protein 6 (PDCD6) expression in CRC samples were analyzed by immunohistochemistry and immunofluorescence. The prognosis between PDCD6 and clinical features were analyzed. The roles of PDCD6 in cellular proliferation and tumor growth were measured by using CCK8, colony formation, and tumor xenograft in nude mice. RNA-sequence (RNA-seq), Mass Spectrum (MS), Co-Immunoprecipitation (Co-IP) and Western blot were utilized to investigate the mechanism of tumor progression. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were performed to determine the correlation of PDCD6 and MAPK pathway. RESULTS: Higher expression levels of PDCD6 in tumor tissues were associated with a poorer prognosis in patients with CRC. Furthermore, PDCD6 increased cell proliferation in vitro and tumor growth in vivo. Mechanistically, RNA-seq showed that PDCD6 could affect the activation of the MAPK signaling pathway. PDCD6 interacted with c-Raf, resulting in the activation of downstream c-Raf/MEK/ERK pathway and the upregulation of core cell proliferation genes such as MYC and JUN. CONCLUSIONS: These findings reveal the oncogenic effect of PDCD6 in CRC by activating c-Raf/MEK/ERK pathway and indicate that PDCD6 might be a potential prognostic indicator and therapeutic target for patients with colorectal cancer.

Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

BACKGROUND: Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS: We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58 days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS: Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS: These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.

Development of a Metastasis-Related Immune Prognostic Model of Metastatic Colorectal Cancer and Its Usefulness to Immunotherapy.

Background: Post-surgical recurrence of the metastatic colorectal cancer (mCRC) remains a challenge, even with adjuvant therapy. Moreover, patients show variable outcomes. Here, we set to identify gene models based on the perspectives of intrinsic cell activities and extrinsic immune microenvironment to predict the recurrence of mCRC and guide the adjuvant therapy. Methods: An RNA-based gene expression analysis of CRC samples (total = 998, including mCRCs = 344, non-mCRCs = 654) was performed. A metastasis-evaluation model (MEM) for mCRCs was developed using the Cox survival model based on the prognostic differentially expressed genes between mCRCs and non-mCRCs. This model separated the mCRC samples into high- and low-recurrence risk clusters that were tested using machine learning to predict recurrence. Further, an immune prognostic model (IPM) was built using the COX survival model with the prognostic differentially expressed immune-related genes between the two MEM risk clusters. The ability of MEM and IPM to predict prognosis was analyzed and validated. Moreover, the IPM was utilized to evaluate its relationship with the immune microenvironment and response to immuno-/chemotherapy. Finally, the dysregulation cause of IPM three genes was analyzed in bioinformatics. Results: A high post-operative recurrence risk was observed owing to the downregulation of the immune response, which was influenced by MEM genes (BAMBI, F13A1, LCN2) and their related IPM genes (SLIT2, CDKN2A, CLU). The MEM and IPM were developed and validated through mCRC samples to differentiate between low- and high-recurrence risk in a real-world cohort. The functional enrichment analysis suggested pathways related to immune response and immune system diseases as the major functional pathways related to the IPM genes. The IPM high-risk group (IPM-high) showed higher fractions of regulatory T cells (Tregs) and smaller fractions of resting memory CD4+ T cells than the IPM-low group. Moreover, the stroma and immune cells in the IPM-high samples were scant. Further, the IPM-high group showed downregulation of MHC class II molecules. Additionally, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and GDSC analysis suggested the IPM-low as a promising responder to anti-CTLA-4 therapy and the common FDA-targeted drugs, while the IPM-high was non-responsive to these treatments. However, treatment using anti-CDKN2A agents, along with the activation of major histocompatibility complex (MHC) class-II response might sensitize this refractory mCRC subgroup. The dysfunction of MEIS1 might be the reason for the dysregulation of IPM genes. Conclusions: The IPM could identify subgroups of mCRC with a distinct risk of recurrence and stratify the patients sensitive to immuno-/chemotherapy. Further, for the first time, our study highlights the importance of MHC class-II molecules in the treatment of mCRCs using immunotherapy.

Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition.

The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage-induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX. SIGNIFICANCE: ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers.See related commentary by Cole, p. 375.

Structural mapping of hot spots within human CASPR2 discoidin domain for autoantibody recognition.

Accumulating evidence has showed that anti-CASPR2 autoantibodies occur in a long list of neurological immune disorders including limbic encephalitis (LE). Belonging to the well-known neurexin superfamily, CASPR2 has been suggested to be a central node in the molecular networks controlling neurodevelopment. Distinct from other subfamilies in the neurexin superfamily, the CASPR subfamily features a unique discoidin (Disc) domain. As revealed by our and others' recent studies, CASPR2 Disc domain bears a major epitope for autoantibodies. However, structural information on CASPR2 recognition by autoantibodies has been lacking. Here, we report the crystal structure of human CASPR2 Disc domain at a high resolution of 1.31 Å, which is the first atomic-resolution structure of the CASPR subfamily members. The Disc domain adopts a total ß structure and folds into a distorted jellyroll-like barrel with a conserved disulfide-bond interlocking its N- and C-termini. Defined by four loops and located in one end of the barrel, the "loop-tip surface" is totally polar and easily available for protein docking. Based on structure-guided epitope prediction, we generated nine mutants and evaluated their binding to autoantibodies of cerebrospinal fluid from twelve patients with limbic encephalitis. The quadruple mutant G69N/A71S/S77N/D78R impaired CASPR2 binding to autoantibodies from eleven LE patients, which indicates that the loop L1 in the Disc domain bears hot spots for autoantibody interaction. Structural mapping of autoepitopes within human CASPR2 Disc domain sheds light on how autoantibodies could sequester CASPR2 ectodomain and antagonize its functionalities in the pathogenic processes.

Structural characterizations of human periostin dimerization and cysteinylation.

Human periostin plays a multifaceted role in remodeling the extracellular matrix milieu by interacting with other proteins and itself in both a heterophilic and homophilic manner. However, the structural mechanism for its extensive interactions has remained elusive. Here, we report the crystal structures of human periostin (EMI-Fas1I-IV ) and its Cys60Ala mutant. In combination with multi-angle light-scattering analysis and biochemical assays, the crystal structures reveal that periostin mainly exists as a dimer in solution and its homophilic interaction is mainly mediated by the EMI domain. Furthermore, Cys60 undergoes cysteinylation as confirmed by mass spectroscopy, and this site hardly affects the homophilic interaction. Also, the structures yield insights into how periostin forms heterophilic interactions with other proteins under physiological or pathological conditions.

Recently Evolved Tumor Suppressor Transcript TP73-AS1 Functions as Sponge of Human-Specific miR-941.

MicroRNA (miRNA) sponges are vital components of posttranscriptional gene regulation. Yet, only a limited number of miRNA sponges have been identified. Here, we show that the recently evolved noncoding tumor suppressor transcript, antisense RNA to TP73 gene (TP73-AS1), functions as a natural sponge of human-specific miRNA miR-941. We find unusually nine high-affinity miR-941 binding sites clustering within 1 kb region on TP73-AS1, which forms miR-941 sponge region. This sponge region displays increased sequence constraint only in humans, and its formation can be traced to the tandem expansion of a 71-nt-long sequence containing a single miR-941 binding site in old world monkeys. We further confirm TP73-AS1 functions as an efficient miR-941 sponge based on massive transcriptome data analyses, wound-healing assay, and Argonaute protein immunoprecipitation experiments conducted in cell lines. The expression of miR-941 and its sponge correlate inversely across multiple healthy and cancerous tissues, with miR-941 being highly expressed in tumors and preferentially repressing tumor suppressors. Thus, the TP73-AS1 and miR-941 duo represents an unusual case of the extremely rapid evolution of noncoding regulators controlling cell migration, proliferation, and tumorigenesis.

Structural basis of SALM5-induced PTPδ dimerization for synaptic differentiation.

SALM5, a synaptic adhesion molecule implicated in autism, induces presynaptic differentiation through binding to the LAR family receptor protein tyrosine phosphatases (LAR-RPTPs) that have been highlighted as presynaptic hubs for synapse formation. The mechanisms underlying SALM5/LAR-RPTP interaction remain unsolved. Here we report crystal structures of human SALM5 LRR-Ig alone and in complex with human PTPδ Ig1-3 (MeA-). Distinct from other LAR-RPTP ligands, SALM5 mainly exists as a dimer with LRR domains from two protomers packed in an antiparallel fashion. In the 2:2 heterotetrameric SALM5/PTPδ complex, a SALM5 dimer bridges two separate PTPδ molecules. Structure-guided mutations and heterologous synapse formation assays demonstrate that dimerization of SALM5 is prerequisite for its functionality in inducing synaptic differentiation. This study presents a structural template for the SALM family and reveals a mechanism for how a synaptic adhesion molecule directly induces cis-dimerization of LAR-RPTPs into higher-order signaling assembly.

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins.

The Wnt-signaling pathway is crucial to cell proliferation, differentiation, and migration. The secreted Frizzled-related proteins (sFRPs) represent the largest family of secreted Wnt inhibitors. However, their function in antagonizing Wnt signaling has remained somewhat controversial. Here, we report the crystal structure of Sizzled from Xenopus laevis, the first full-length structure of an sFRP. Tethered by an inter-domain disulfide bond and a linker, the N-terminal cysteine-rich domain (CRD) and the C-terminal netrin-like domain (NTR) of Sizzled are arranged in a tandem fashion, with the NTR domain occluding the groove of CRD for Wnt accessibility. A Dual-Luciferase assay demonstrated that removing the NTR domain and replacing the CRD groove residues His-116 and His-118 with aromatic residues may significantly enhance antagonistic function of Sizzled in inhibiting Wnt3A signaling. Sizzled is a monomer in solution, and Sizzled CRD exhibited different packing in the crystal, suggesting that sFRPs do not have a conserved CRD dimerization mode. Distinct from the canonical NTR domain, the Sizzled NTR adopts a novel α/ß folding with two perpendicular helices facing the central mixed ß-sheet. The subgroup of human sFRP1/2/5 and Sizzled should have a similar NTR domain that features a highly positively charged region, opposite the NTR-CRD interface, suggesting that the NTR domain in human sFRPs, at least sFRP1/2/5, is unlikely to bind to Wnt but is likely involved in biphasic Wnt signaling modulation. In summary, the Sizzled structure provides the first insights into how the CRD and the NTR domains relate to each other for modulating Wnt-antagonistic function of sFRPs.