PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

BTB domains: A structural view of evolution, multimerization, and protein-protein interactions.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) is a conserved domain found in many eukaryotic proteins with diverse cellular functions. Recent studies revealed its importance in multiple developmental processes as well as in the onset and progression of oncological diseases. Most BTB domains can form multimers and selectively interact with non-BTB proteins. Structural studies of BTB domains delineated the presence of different interfaces involved in various interactions mediated by BTBs and provided a basis for the specific inhibition of distinct protein-interaction interfaces. BTB domains originated early in eukaryotic evolution and progressively adapted their structural elements to perform distinct functions. In this review, we summarize and discuss the structural principles of protein-protein interactions mediated by BTB domains based on the recently published structural data and advances in protein modeling. We propose an update to the structure-based classification of BTB domain families and discuss their evolutionary interconnections.

Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo- DNA Polymerase.

Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo- is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo- binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the "closed" form of Bst exo- to the purine-rich DNA sequences, especially those containing dG at the 3'-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3'- and 5'-terminal motifs. It has been shown that templates with the oligopurine 3'-terminal fragment and oligopyrimidine 5'-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

The Oligomeric State of Vasorin in the Plasma Membrane Measured Non-Invasively by Quantitative Fluorescence Fluctuation Spectroscopy.

Vasorin (VASN), a transmembrane protein heavily expressed in endothelial cells, has garnered recent interest due to its key role in vascular development and pathology. The oligomeric state of VASN is a crucial piece of knowledge given that receptor clustering is a frequent regulatory mechanism in downstream signaling activation and amplification. However, documentation of VASN oligomerization is currently absent. In this brief report, we describe the measurement of VASN oligomerization in its native membranous environment, leveraging a class of fluorescence fluctuation spectroscopy. Our investigation revealed that the majority of VASN resides in a monomeric state, while a minority of VASN forms homodimers in the cellular membrane. This result raises the intriguing possibility that ligand-independent clustering of VASN may play a role in transforming growth factor signaling.

Phosphatidylserine exposure modulates adhesion GPCR BAI1 (ADGRB1) signaling activity.

Brain-specific angiogenesis inhibitor 1 (BAI1; also called ADGRB1 or B1) is an adhesion G protein-coupled receptor known from studies on macrophages to bind to phosphatidylserine (PS) on apoptotic cells via its N-terminal thrombospondin repeats. A separate body of work has shown that B1 regulates postsynaptic function and dendritic spine morphology via signaling pathways involving Rac and Rho. However, it is unknown if PS binding by B1 has any effect on the receptor's signaling activity. To shed light on this subject, we studied G protein-dependent signaling by B1 in the absence and presence of coexpression with the PS flippase ATP11A in human embryonic kidney 293T cells. ATP11A expression reduced the amount of PS exposed extracellularly and also strikingly reduced the signaling activity of coexpressed full-length B1 but not a truncated version of the receptor lacking the thrombospondin repeats. Further experiments with an inactive mutant of ATP11A showed that the PS flippase function of ATP11A was required for modulation of B1 signaling. In coimmunoprecipitation experiments, we made the surprising finding that ATP11A not only modulates B1 signaling but also forms complexes with B1. Parallel studies in which PS in the outer leaflet was reduced by an independent method, deletion of the gene encoding the endogenous lipid scramblase anoctamin 6 (ANO6), revealed that this manipulation also markedly reduced B1 signaling. These findings demonstrate that B1 signaling is modulated by PS exposure and suggest a model in which B1 serves as a PS sensor at synapses and in other cellular contexts.

Targeting GD2-Positive Tumor Cells by Pegylated scFv Fragment-Drug Conjugates Carrying Maytansinoids DM1 and DM4.

Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide-thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol-maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv-PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv-PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment-drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment-drug conjugates for cancer treatment.

New method for microRNA detection based on multimerization.

Detection of specific microRNA (miRNA) is of great demand due to their essential role in genes regulation, stress response and development of diseases. However, mature miRNAs are small molecules that make it difficult to use routine amplification-based methods. Here, we report an approach for detection of miRNA based on a new type of isothermal amplification, namely, multimerization. The proposed technique is simple and versatile, excludes a reverse transcription step, and requires two conventional primers only and no additional stem-loop or fluorogenic probes. Only mature miRNAs can initiate multimerization, thereby, pri- or pre-miRNA are excluded from analysis, ensuring high accuracy of the assay. The approach was approved on miRNA from common wheat Triticum aestivum; the increase of Tae-miRNA159 level for plants affected by Stagonospora nodorum Berk infection was demonstrated. The obtained results allow to perform quantitative analysis, providing determination of specific targets with high reliability (detection limit of about 20 pM).

Heterodimerization With 5-HT2BR Is Indispensable for ß2AR-Mediated Cardioprotection.

RATIONALE: The ß2-adrenoceptor (ß2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the ß2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac ß2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. OBJECTIVE: Here, we aim to investigate the potential cardioprotective effect of ß2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of ß2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). METHODS AND RESULTS: Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the ß2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another ß2-agonist zinterol markedly promoted heterodimerization of ß2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and ß2-ARs enhanced ß2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated ß2-AR-stimulated Gi signaling and cardioprotection. CONCLUSIONS: These data demonstrate that the ß2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of ß2-ARs and 5-HT2BRs.

Detection of Specific RNA Targets by Multimerization.

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to essential role of RNA in transfer of genetic information and development of diseases. Here, we report on an approach for detection of RNA targets based on the particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase, and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was verified by using genetic material of the SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate the SARS-CoV-2 RNA-positive samples from the SARS-CoV-2 negative samples with high reliability. The proposed technique allows detection of RNA even in the samples, which were subjected to multiple freezing-thawing cycles.

Increase in cysteine-mediated multimerization under attractive protein-protein interactions.

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein-protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein-protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

Identification of the Fc-alpha/mu receptor in Xenopus provides insight into the emergence of the poly-Ig receptor (pIgR) and mucosal Ig transport.

The polyimmunoglobulin receptor (pIgR) transcytoses J chain-containing antibodies through mucosal epithelia. In mammals, two cis-duplicates of PIGR, FCMR, and FCAMR, flank the PIGR gene. A PIGR duplication is first found in amphibians, previously annotated as PIGR2 (herein xlFCAMR), and is expressed by APCs. We demonstrate that xlFcamR is the equivalent of mammalian FcamR. It has been assumed that pIgR is the oldest member of this family, yet our data could not distinguish whether PIGR or FCAMR emerged first; however, FCMR was the last family member to emerge. Interestingly, bony fish "pIgR" is not an orthologue of tetrapod pIgR, and possibly acquired its function via convergent evolution. PIGR/FCAMR/FCMR are members of a larger superfamily, including TREM, CD300, and NKp44, which we name the "double-disulfide Ig superfamily" (ddIgSF). Domains related to each ddIgSF family were identified in cartilaginous fish (sharks, chimeras) and encoded in a single gene cluster syntenic to the human pIgR locus. Thus, the ddIgSF families date back to the earliest antibody-based adaptive immunity, but apparently not before. Finally, our data strongly suggest that the J chain arose in evolution only for Ig multimerization. This study provides a framework for further studies of pIgR and the ddIgSF in vertebrates.

NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization.

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine.

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

Inhibition of nonspecific polymerase activity using Poly(Aspartic) acid as a model anionic polyelectrolyte.

DNA polymerases with strand-displacement activity allow to amplify nucleic acids under isothermal conditions but often lead to undesirable by-products. Here, we report the increase of specificity of isothermal amplification in the presence of poly (aspartic) acids (pAsp). We hypothesized that side reactions occur due to the binding of the phosphate backbone of synthesized DNA strands with surface amino groups of the polymerase, and weakly acidic polyelectrolytes could shield polymerase molecules from DNA and thereby inhibit nonspecific amplification. Suppression of nonspecific polymerase activity by pAsp was studied on multimerization as a model side reaction. It was found that a low concentration of pAsp (0.01%) provides successful amplification of specific DNA targets. The inhibitory effect of pAsp is due to its polymeric structure since aspartic acid did affect neither specific nor nonspecific amplification. Strongly acidic polyelectrolyte heparin does not possess the same selectivity since it suppresses any DNA synthesis. The applicability of pAsp to prevent nonspecific reactions and reliable detection of the specific target has been demonstrated on the genetic material of SARS-CoV-2 coronavirus using Loop-mediated isothermal amplification.

Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

The disulfide bond Cys2724-Cys2774 in the C-terminal cystine knot domain of von Willebrand factor is critical for its dimerization and secretion.

BACKGROUND: Type 3 von Willebrand disease (VWD) exhibits severe hemorrhagic tendency with complicated pathogenesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, however, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear. METHOD AND RESULTS: In this study, by the next-generation sequencing we found a missense mutation a c.8171G>A (C2724Y) in the CTCK domain of VWF allele in a patient family with type 3 VWD. In vitro, VWF C2724Y protein was expressed normally in HEK-293T cells but did not form a dimer or secrete into cell culture medium, suggesting that C2724 is critical for the VWF dimerization, and thus for VWF multimerization and secretion. CONCLUSIONS: Our findings provide the first genetic evidence for the important role of Cys2724-Cys2774 in VWF biosynthesis and secretion. Therefore, all of the four intrachain disulfide bonds in CTCK monomer contribute to VWF dimerization and secretion.

TMEM59 potentiates Wnt signaling by promoting signalosome formation.

Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.