Homodimer Formation Research Articles

66 (PB056) - Anti-tumor activity of BDTX-1535, an irreversible CNS penetrant inhibitor of multiple EGFR extracellular domain alterations, in preclinical glioblastoma models

Background: EGFR genetic alterations, including mutation and amplification, are very common in glioblastoma multiforme (GBM), however, no targeted therapy has been approved for this disease. EGFR oncogenic mutations in GBM promote EGFR oncogenic covalent homodimer formation which leads to paradoxical activation and renders reversible inhibitors ineffective. An effective EGFR inhibitor in GBM must meet four conditions:1Highly potent and selective against a broad range of extracellular domain mutations and amplified EGFR,2Wild-type EGFR sparing,3Irreversible binding to circumvent paradoxical activation,4High CNS penetration. Our MAP discovery platform led to the identification of MasterKey inhibitor BDTX-1535, an irreversible 4th-generation CNS penetrant EGFR tyrosine kinase inhibitor (TKI) that potently and selectively targets a broad range of EGFR alterations in GBM. Materials and Methods: BDTX-1535 preclinical exposure was evaluated in multiple species in plasma and brain. Antitumor activity was assessed across a broad range of mouse PDX and allograft models, including an intracranial PDX. 2540 tumors were sequenced with the Tempus xT NGS assay. Mutational frequencies and co-expression status of oncogenic EGFR variants were assessed. The relationship between treatment history and oncogenic EGFR mutational frequency was analyzed. Results: BDTX-1535 is a small molecule that irreversibly inhibits a spectrum of EGFR alterations expressed in GBM together with EGFR amplification, while sparing inhibition of normally expressed wild type EGFR. BDTX-1535 avoids efficacy limiting paradoxical activation that can occur with reversible EGFR inhibitors. BDTX-1535 exhibits good oral bioavailability and a CNS Kpuu of 0.55 and 0.48 in rat and dog, respectively, comparing favorably with other CNS penetrant TKIs. Tumor regression and survival advantage were observed across all tumor models with EGFR amplification or mutation, including in an intracranial PDX model. EGFR mutations and amplification are commonly expressed in GBM. Consistent with published data, EGFR mutations, variants, and copy number gain were frequently co-expressed. For example, among tumors expressing EGFRvIII, ∼95% co-expressed at least one other variant, and among tumors expressing EGFRvVI, ∼45% co-expressed at least one other variant. Within a subset of GBM patients with longitudinal sequencing data, oncogenic EGFR mutations were expressed and persisted following front-line treatment. Conclusions: BDTX-1535 is a potent, CNS penetrant, irreversible small molecule EGFR inhibitor that shows efficacy against a broad range of extracellular domain mutations and EGFR amplification found in GBM patients. RWE findings demonstrate a heterogeneous EGFR mutational profile in GBM patients, with persistent prevalence over time. BDTX-1535 is currently in a phase I clinical study (NCT05256290). Conflict of interest: Ownership: Black Diamond Therapeutics Tempus Labs

Staphylococcus aureus Exfoliative Toxin E, Oligomeric State and Flip of P186: Implications for Its Action Mechanism.

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST2 and SST5 homo- and hetero-dimer formation.

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)–based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P < 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P < 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulates SST2 and SST5 intracellular trafficking induced by octreotide and pasireotide.

Atypical IκB Bcl3 enhances the generation of the NF-κB p52 homodimer

The NF-κB family of dimeric transcription factors regulate diverse biological functions. Their cellular expression profiles differ, which lead to different concentrations in different cell/tissue types. Although the activation mechanisms of different NF-κB dimers have been widely investigated, there is limited information on specific NF-κB dimers’ formation. The NF-κB p52:p52 homodimer regulates an important subset of target genes in cancer cells; however, the molecular mechanism of the generation of this specific homodimer remains unclear. Our study has revealed that the atypical IκB protein, Bcl3, plays an essential role in enhancing the p52:p52 homodimer population which is a unique mechanism to p52 within the NF-κB family. p52 was shown to heterodimerize with four other NF-κB subunits (RelA, RelB, cRel, and p50); all heterodimers, except p52:p50, are significantly more stable than the p52:p52 homodimer. Bcl3 is able to compete with all other NF-κB subunits in cells for efficient p52:p52 homodimer formation which consequently leads to the upregulation of target genes that are involved in cell proliferation, migration, and inflammation, which explain why aberrant activation of Bcl3 and p52 leads to cancer.

A nematode effector inhibits plant immunity by preventing cytosolic free Ca2+ rise.

The Meloidogyne enterolobii effector MeTCTP is a member of the translationally controlled tumour protein (TCTP) family, involved in M. enterolobii parasitism. In this study, we found that MeTCTP forms homodimers and, in this form, binds calcium ions (Ca2+ ). At the same time, Ca2+ could induce homodimerization of MeTCTP. We further identified that MeTCTP inhibits the increase of cytosolic free Ca2+ concentration ([Ca2+ ]cyt ) in plant cells and suppresses plant immune responses. This includes suppression of reactive oxygen species burst and cell necrosis, further promoting M. enterolobii parasitism. Our results have elucidated that the effector MeTCTP can directly target Ca2+ by its homodimeric form and prevent [Ca2+ ]cyt rise in plant roots, revealing a novel mechanism utilized by plant pathogens to suppress plant immunity.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein–protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer’s RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and V max of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

A peptide interfering with the dimerization of oncogenic KITENIN protein and its stability suppresses colorectal tumour progression.

The stability of a protein, as well as its function and versatility, can be enhanced through oligomerization. KITENIN (KAI1 C‐terminal interacting tetraspanin) is known to promote the malignant progression of colorectal cancer (CRC). How KITENIN maintains its structural integrity and stability are largely unknown, however. Here we investigated the mechanisms regulating the stability of KITENIN with the aim of developing therapeutics blocking its oncogenic functions. We found that KITENIN formed a homo‐oligomeric complex and that the intracellular C‐terminal domain (KITENIN‐CTD) was needed for this oligomerization. Expression of the KITENIN‐CTD alone interfered with the formation of the KITENIN homodimer, and the amino acid sequence from 463 to 471 within the KITENIN‐CTD was the most effective. This sequence coupled with a cell‐penetrating peptide was named a KITENIN dimerization‐interfering peptide (KDIP). We next studied the mechanisms by which KDIP affected the stability of KITENIN. The KITENIN‐interacting protein myosin‐X (Myo10), which has oncogenic activity in several cancers, functioned as an effector to stabilize the KITENIN homodimer in the cis formation. Treatment with KDIP resulted in the disintegration of the homodimer via downregulation of Myo10, which led to increased binding of RACK1 to the exposed RACK1‐interacting motif (463–471 aa), and subsequent autophagy‐dependent degradation of KITENIN and reduced CRC cell invasion. Intravenous injection of KDIP significantly reduced the tumour burden in a syngeneic mouse tumour model and colorectal liver metastasis in an intrasplenic hepatic metastasis model. Collectively, our present results provide a new cancer therapeutic peptide for blocking colorectal liver metastasis, which acts by inducing the downregulation of Myo10 and specifically targeting the stability of the oncogenic KITENIN protein.

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2.

The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.

Abstract CT248: Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors

Abstract Background: BRAFV600 alterations (BRAF Class (Cl) I alterations) encode a highly active monomeric oncogenic kinase that can be targeted by approved RAF kinase inhibitors (RAFi) in patients (pts) with melanoma (MEL), NSCLC, colorectal &amp; anaplastic thyroid cancers. Cl II and III represent molecularly distinct BRAF alterations driving dimer-dependent signaling. Cl II alterations include single nucleotide variants (SNVs), indels and gene fusions that induce BRAF homodimer formation, leading to oncogenic kinase activation. Cl III alterations, typically SNVs, are low activity alterations that promote RAF dimerization and RAS-dependent oncogenic MAPK pathway signaling. Approved BRAFi have limited clinical activity in solid tumors driven by BRAF Cl II or III alterations, highlighting the urgency to develop effective targeted therapies for these patients. Combinations of a RAFi and a MEK inhibitor (MEKi) show promising results in early clinical trials. KIN-2787 is a potent, highly selective type II pan-RAFi designed to target both monomeric &amp; dimeric forms of the altered BRAF kinase and minimize paradoxical activation, a liability often observed with other RAFi that can adversely impact tolerability &amp; require addition of a MEKi to suppress pathway activation. KIN-2787 has favorable pharmaceutical properties, achieves substantial systemic exposures in pre-clinical toxicology studies, &amp; induces regressions in human cancer xenograft models driven by BRAF Cl I, II, or III alterations. Combinations of KIN-2787 with a MEKi show promising activity in preclinical models. Methods: This is a FIH, multicenter, non-randomized Ph1 study (NCT04913285) of KIN-2787 in adult pts with BRAF mutant advanced &amp; metastatic solid tumors (AMST). KIN-2787 will be given orally bid continuously in 28-day cycles until drug intolerance or disease progression. Planned sample size is approx. 155 pts in 2 parts: Part A is a dose-escalation to MTD open to pts with AMST driven by either BRAF genomic alterations (Cl I, II or III ) or by NRAS. Part A1 assesses single agent KIN-2787; Part A2 assesses KIN-2787 in combination w/binimetinib. Part B will evaluate a selected dose of KIN-2787 in 3 cohorts of pts (MEL, NSCLC, or other AMST), each driven by BRAF Cl II or III alterations. Standard Ph1 enrollment criteria are required (ECOG PS 0-2, normal organ function, prior receipt of standard treatment or medical judgment that such is not appropriate). Pts may have measurable or evaluable disease. Key exclusion criteria include known active brain metastases, prior BRAF-, MEK-, or MAPK inhibitor therapy (unless for FDA approved indications), HBV/HCV seropositivity. Primary endpoints are safety/tolerability (Part A), and preliminary antitumor activity: objective response rate, disease control rate, duration of response, &amp; duration of stable disease (Part B). Secondary objectives include pharmacokinetic and pharmacodynamic assessments including measures of MAPK signaling inhibition. Enrollment began in August 2022. Trial is open globally. Citation Format: Meredith McKean, Cesar A. Perez, Shumei Kato, Alexander I. Spira, Yan Xing, Hao Xie, Michael Millward, Anthony Olszanski, Ken Kobayashi, Nichol Miller, Paul Severson, Betty Tam, Caro Unger. Design and rationale of a first in human (FIH) phase 1/1b study evaluating KIN-2787, a potent and highly selective pan-RAF inhibitor, in adult patients with BRAF- and NRAS-mutation positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT248.

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERβ in pancreatic beta cells

17β-estradiol protects pancreatic β-cells from apoptosis via the estrogen receptors ERα, ERβ and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERβ, or in dispersed islet cells from ERβ knockout (BERKO) mice. However, the ERα and ERβ agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERβ form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERβ as well as GPER activation by G1 decreased ERαβ heterodimers. We propose that ERαβ heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαβ heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

HSP90 N-terminal inhibitors target oncoprotein MORC2 for autophagic degradation and suppress MORC2-driven breast cancer progression.

AimsMORC family CW‐type zinc finger 2 (MORC2), a GHKL‐type ATPase, is aberrantly upregulated in multiple types of human tumors with profound effects on cancer aggressiveness, therapeutic resistance, and clinical outcome, thus making it an attractive drug target for anticancer therapy. However, the antagonists of MORC2 have not yet been documented.Methods and ResultsWe report that MORC2 is a relatively stable protein, and the N‐terminal homodimerization but not ATP binding and hydrolysis is crucial for its stability through immunoblotting analysis and Quantitative real‐time PCR. The N‐terminal but not C‐terminal inhibitors of heat shock protein 90 (HSP90) destabilize MORC2 in multiple cancer cell lines, and strikingly, this process is independent on HSP90. Mechanistical investigations revealed that HSP90 N‐terminal inhibitors disrupt MORC2 homodimer formation without affecting its ATPase activities, and promote its lysosomal degradation through the chaperone‐mediated autophagy pathway. Consequently, HSP90 inhibitor 17‐AAG effectively blocks the growth and metastatic potential of MORC2‐expressing breast cancer cells both in vitro and in vivo, and these noted effects are not due to HSP90 inhibition.ConclusionWe uncover a previously unknown role for HSP90 N‐terminal inhibitors in promoting MORC2 degradation in a HSP90‐indepentent manner and support the potential application of these inhibitors for treating MORC2‐overexpressing tumors, even those with low or absent HSP90 expression. These results also provide new clue for further design of novel small‐molecule inhibitors of MORC2 for anticancer therapeutic application.

Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C−H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co‐synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non‐heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

The Role of N-Glycosylation in the Intracellular Trafficking and Functionality of Neuronal Growth Regulator 1.

Neuronal growth regulator 1 (NEGR1) is a brain-enriched membrane protein that is involved in neural cell communication and synapse formation. Accumulating evidence indicates that NEGR1 is a generic risk factor for various psychiatric diseases including autism and depression. Endoglycosidase digestion of single NEGR1 mutants revealed that the wild type NEGR1 has six putative N-glycosylation sites partly organized in a Golgi-dependent manner. To understand the role of each putative N-glycan residue, we generated a series of multi-site mutants (2MT–6MT) with additive mutations. Cell surface staining and biotinylation revealed that NEGR1 mutants 1MT to 4MT were localized on the cell surface at different levels, whereas 5MT and 6MT were retained in the endoplasmic reticulum to form highly stable multimer complexes. This indicated 5MT and 6MT are less likely to fold correctly. Furthermore, the removal of two N-terminal sites N75 and N155 was sufficient to completely abrogate membrane targeting. An in vivo binding assay using the soluble NEGR1 protein demonstrated that glycans N286, N294 and N307 on the C-terminal immunoglobulin-like domain play important roles in homophilic interactions. Taken together, these results suggest that the N-glycan moieties of NEGR1 are closely involved in the folding, trafficking, and homodimer formation of NEGR1 protein in a site-specific manner.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Novel Loss-of-Function Mutations in NPR2 Cause Acromesomelic Dysplasia, Maroteaux Type.

Acromesomelic dysplasia, Maroteaux type (AMDM) is a rare skeletal dysplasia characterized by severe disproportionate short stature, short hands and feet, normal intelligence, and facial dysmorphism. Homozygous or compound heterozygous mutations in the natriuretic peptide receptor 2 (NPR2) gene produce growth-restricted phenotypes. The current study was designed to identify and characterize NPR2 loss-of-function mutations in patients with AMDM and to explore therapeutic responses to recombinant growth hormone (rhGH). NPR2 was sequenced in two Chinese patients with AMDM via next generation sequencing, and in silico structural analysis or transcript analysis of two novel variants was performed to examine putative protein changes. rhGH treatment was started for patient 1. Three NPR2 mutations were identified in two unrelated cases: two compound heterozygous mutations c.1112G>A p.(Arg371Gln) and c.2887+2T>C in patient 1 and a homozygous mutation c.329G>A p.(Arg110His) in patient 2, yielding distinct phenotypes. RNA extracted from peripheral blood cells of patient 1 showed alternatively spliced transcripts not present in control cells. Homology modeling analyses suggested that the c.1112G>A p.(Arg371Gln) mutation disrupted the binding of NPR-B homodimer to its ligand (C-type natriuretic peptide) in the extracellular domain as a result of global allosteric effects on homodimer formation. Thus, c.2887+2T>C and c.1112G>A p.(Arg371Gln) in NPR2 were loss-of-function mutations. Furthermore, rhGH therapy in patient 1 increased the patient’s height by 0.6SDS over 15 months without adversely affecting the trunk-leg proportion. The short-term growth-promoting effect was equivalent to that reported for idiopathic short stature. Overall, our findings broadened the genotypic spectrum of NPR2 mutations in individuals with AMDM and provided insights into the efficacy of rhGH in these patients.

Self-Assembly of Intracellular Multivalent RNA Complexes Using Dimeric Corn and Beetroot Aptamers.

DNA and RNA can spontaneously self-assemble into various structures, including aggregates, complexes, and ordered structures. The self-assembly reactions cannot be genetically encoded to occur in living mammalian cells since the double-stranded nucleic acids generated by current self-assembly approaches are unstable and activate innate RNA immunity pathways. Here, we show that recently described dimeric aptamers can be used to create RNAs that self-assemble and create RNA and RNA-protein assemblies in cells. We find that incorporation of five copies of Corn, a dimeric fluorogenic RNA aptamer, into an RNA causes the RNA to form large clusters in cells, reflecting multivalent RNA-RNA interactions enabled by these RNAs. Here, we also describe a second dimeric fluorogenic aptamer, Beetroot, which shows partial sequence similarity to Corn. Both Corn and Beetroot form homodimers with themselves but do not form Corn-Beetroot heterodimers. We thus use Corn and Beetroot to encode distinct RNA-protein assemblies in the same cells. Overall, these studies provide an approach for inducing RNA self-assembly, enable multiplexing of distinct RNA assemblies in cells, and demonstrate that proteins can be recruited to RNA assemblies to genetically encode intracellular RNA-protein assemblies.

A functionally divergent SOC1 homolog improves soybean yield and latitudinal adaptation

Soybean (Glycine max) grows in a wide range of latitudes, but it is extremely sensitive to photoperiod, which reduces its yield and ability to adapt to different environments. Therefore, understanding of the genetic basis of soybean adaptation is of great significance for breeding and improvement. Here, we characterized Tof18 (SOC1a) that conditions early flowering and growth habit under both short-day and long-day conditions. Molecular analysis confirmed that the two SOC1 homologs present in soybeans (SOC1a and SOC1b) underwent evolutionary functional divergence, with SOC1a having stronger effects on flowering time and stem node number than SOC1b due to transcriptional differences. soc1a soc1b double mutants showed stronger functional effects than either of the single mutants, perhaps due to the formation of SOC1a and SOC1b homodimers or heterodimers. Additionally, Tof18/SOC1a improves the latitudinal adaptation of cultivated soybeans, highlighting the functional importance of SOC1a. The Tof18G allele facilitates adaptation to high latitudes, whereas Tof18A facilitates adaptation to low latitudes. We demonstrated that SOC1s contribute to floral induction in both leaves and shoot apex through inter-regulation with FTs. The SOC1a-SOC1b-Dt2 complex plays essential roles in stem growth habit by directly binding to the regulatory sequence of Dt1, making the genes encoding these proteins potential targets for genome editing to improve soybean yield via molecular breeding. Since the natural Tof18A allele increases node number, introgressing this allele into modern cultivars could improve yields, which would help optimize land use for food production in the face of population growth and global warming.