Structural basis for antiepileptic drugs and botulinum neurotoxin recognition of SV2A.

More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. Synaptic vesicle glycoprotein 2a (SV2A), a putative membrane transporter in the synaptic vesicles (SVs), has been identified as a target of LEV and BRV. SV2A also serves as a receptor for botulinum neurotoxin (BoNT), which is the most toxic protein and has paradoxically emerged as a potent reagent for therapeutic and cosmetic applications. Nevertheless, no structural analysis on AEDs and BoNT recognition by full-length SV2A has been available. Here we describe the cryo-electron microscopy structures of the full-length SV2A in complex with the BoNT receptor-binding domain, BoNT/A2 HC, and either LEV or BRV. The large fourth luminal domain of SV2A binds to BoNT/A2 HC through protein-protein and protein-glycan interactions. LEV and BRV occupy the putative substrate-binding site in an outward-open conformation. A propyl group in BRV creates additional contacts with SV2A, explaining its higher binding affinity than that of LEV, which was further supported by label-free spectral shift assay. Numerous LEV derivatives have been developed as AEDs and positron emission tomography (PET) tracers for neuroimaging. Our work provides a structural framework for AEDs and BoNT recognition of SV2A and a blueprint for the rational design of additional AEDs and PET tracers.

Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

Synthetic modulators for plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Here, we describe a novel small-molecule inhibitor for 14-3-3 proteins ofArabidopsis thaliana. The inhibitor was identified from unexpected products in DMSO stock solution of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assaysrevealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide a new insight into the design of potent and isoform-selective 14-3-3 modulators.

Remodeling of the postsynaptic proteome in male mice and marmosets during synapse development.

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric müllerian duct development by eliminating asymmetric gene expression in japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Rationalizing the Influence of the Binding Affinity on the Activity of Phosphoserine Phosphatases.

The Sabatier principle states that catalytic activity can be maximized when the substrate binding affinity is neither too strong nor too weak. Recent studies have shown that the activity of several hydrolases is maximized at intermediate values of the binding affinity (Michaelis-Menten constant: Km ). However, it remains unclear whether this concept of artificial catalysis is applicable to enzymes in general, especially for those which have evolved under different reaction environments. Herein, we show that the activity of phosphoserine phosphatase is also enhanced at an intermediate Km value of approximately 0.5 mM. Within our dataset, the variation of Km by three orders of magnitude accounted for a roughly 18-fold variation in the activity. Owing to the high phylogenetic and physiological diversity of our dataset, our results support the importance of optimizing Km for enzymes in general. On the other hand, a 77-fold variation in the activity was attributed to other physicochemical parameters, such as the Arrhenius prefactor of kcat , and could not be explained by the Sabatier principle. Therefore, while tuning the binding affinity according to the Sabatier principle is an important consideration, the Km value is only one of many physicochemical parameters which must be optimized to maximize enzymatic activity.

Attenuation of protein arginine dimethylation via S-nitrosylation of protein arginine methyltransferase 1.

Upregulation of nitric oxide (NO) production contributes to the pathogenesis of numerous diseases via S-nitrosylation, a post-translational modification of proteins. This process occurs due to the oxidative reaction between NO and a cysteine thiol group; however, the extent of this reaction remains unknown. S-Nitrosylation of PRMT1, a major asymmetric arginine methyltransferase of histones and numerous RNA metabolic proteins, was induced by NO donor treatment. We found that nitrosative stress leads to S-nitrosylation of cysteine 119, located near the active site, and attenuates the enzymatic activity of PRMT1. Interestingly, RNA sequencing analysis revealed similarities in the changes in expression elicited by NO and PRMT1 inhibitors or knockdown. A comprehensive search for PRMT1 substrates using the proximity-dependent biotin identification method highlighted many known and new substrates, including RNA-metabolizing enzymes. To validate this result, we selected the RNA helicase DDX3 and demonstrated that arginine methylation of DDX3 is induced by PRMT1 and attenuated by NO treatment. Our results suggest the existence of a novel regulatory system associated with transcription and RNA metabolism via protein S-nitrosylation.

Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor.

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Successful Pregnancy Outcome in a Patient with Juvenile Idiopathic Arthritis in Adulthood, Amyloid A Amyloidosis, and Chronic Kidney Disease Using Tocilizumab with Strict Blood Pressure Control.

Pregnancies with chronic kidney disease (CKD) and high disease activity in rheumatic diseases are high-risk events with adverse outcomes for both the mother and fetus. We herein report a 35-year-old woman with juvenile idiopathic arthritis (JIA), amyloid A (AA) amyloidosis related to JIA, and CKD stage G4A2 who wished to have children. She achieved a successful pregnancy, even in the presence of these multiple risk factors, using tocilizumab to control the disease activity of JIA and AA amyloidosis, along with antihypertensive drugs to control her blood pressure before and during pregnancy.

Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

Urinary growth differentiation factor 15 predicts renal function decline in diabetic kidney disease.

Sensitive biomarkers can enhance the diagnosis, prognosis, and surveillance of chronic kidney disease (CKD), such as diabetic kidney disease (DKD). Plasma growth differentiation factor 15 (GDF15) levels are a novel biomarker for mitochondria-associated diseases; however, it may not be a useful indicator for CKD as its levels increase with declining renal function. This study explores urinary GDF15's potential as a marker for CKD. The plasma and urinary GDF15 as well as 15 uremic toxins were measured in 103 patients with CKD. The relationship between the urinary GDF15-creatinine ratio and the uremic toxins and other clinical characteristics was investigated. Urinary GDF15-creatinine ratios were less related to renal function and uremic toxin levels compared to plasma GDF15. Additionally, the ratios were significantly higher in patients with CKD patients with diabetes (p = 0.0012) and reduced with statin treatment. In a different retrospective DKD cohort study (U-CARE, n = 342), multiple and logistic regression analyses revealed that the baseline urinary GDF15-creatinine ratios predicted a decline in estimated glomerular filtration rate (eGFR) over 2 years. Compared to the plasma GDF15 level, the urinary GDF15-creatinine ratio is less dependent on renal function and sensitively fluctuates with diabetes and statin treatment. It may serve as a good prognostic marker for renal function decline in patients with DKD similar to the urine albumin-creatinine ratio.

Biogenesis of fibrils requires C-mannosylation of PMEL.

Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.

Gestational arsenic exposure induces anxiety-like behaviors in F1 female mice by dysregulation of neurological and immunological markers.

BACKGROUND: Arsenic is a harmful heavy metal and a well-known developmental neurotoxicant. Previously, we have reported that gestational arsenic exposure resulted in impaired social behaviors in F1 and F2 male mice. However, little is known about the developmental arsenic exposure on anxiety-like behavior. This study aimed to detect the effect of gestational arsenic exposure on anxiety-like behavior and related gene expressions in 74-week-old F1 female mice. METHOD: Pregnant C3H/HeN mice (F0) were given drinking water containing 85 ppm sodium arsenite (NaAsO2) from gestational day 8 to 18. The control mice were given tap water only. At 74-week-old, open field test was performed, then anxiety and apoptosis-related factors were determined by real_time RT_PCR and immunohistochemical analyses. RESULTS: The arsenite-exposed F1 female mice showed decreased center entry and center time in open field test. In addition, the number of grooming and fecal pallet was significantly increased in the arsenite-exposed F1 female mice compared to the control. Downregulation of brain-derived neurotrophic factor (BDNF), serotonin receptor (5HT1A) and upregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), interleukin 1 ß (IL-1ß), cyclooxygenase 2 (COX2), caspase-3, Bcl2-associated X protein (Bax) were detected in the prefrontal cortex in the arsenite-exposed F1 female mice. Microglial marker ionized calcium-binding adapter molecule 1 (Iba1)-positive cells were increased in the arsenite-exposed F1 female mice. Moreover, a significantly increased plasma corticosterone level was observed in the arsenic-exposed F1 female mice. CONCLUSIONS: This study suggested that gestational arsenic exposure induced anxiety-like behavior accompanied with dysregulation of neurological and immunological markers, neuroinflammatory responses, neuronal apoptosis, and decreased neurogenesis in the prefrontal cortex of F1 female mice.

Epigenetic mechanisms to propagate histone acetylation by p300/CBP.

Histone acetylation is important for the activation of gene transcription but little is known about its direct read/write mechanisms. Here, we report cryogenic electron microscopy structures in which a p300/CREB-binding protein (CBP) multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for reading, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 writes by reading H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP replicates histone N-terminal tail acetylation within the H3-H4 tetramer to inherit epigenetic storage, and transcribes it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.

Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells.

Mannose has anticancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anticancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anticancer activity of mannose.

In order to grow and divide, cells require a variety of sugars. Breaking down sugars provides energy for cells to proliferate and allows them to make more complex molecules, such as DNA. Although this principle also applies to cancer cells, a specific sugar called mannose not only inhibits cancer cell division but also makes them more sensitive to chemotherapy. These anticancer effects of mannose are particularly strong in cells lacking a protein known as MPI, which breaks down mannose. Evidence from honeybees suggests that a combination of mannose and low levels of MPI leads to a build-up of a modified form of mannose, called mannose-6-phosphate, within cells. As a result, pathways required to release energy from glucose become disrupted, proving lethal to these insects. However, it was not clear whether the same processes were responsible for the anticancer effects of mannose. To investigate, Harada et al. removed the gene that encodes the MPI protein in two types of human cancer cells. The experiments showed that mannose treatment was not lethal to these cells but overall slowed the cell cycle ­ a fundamental process for cell growth and division. More detailed biochemical experiments showed that cancer cells with excess mannose-6-phosphate could not produce the molecules required to make DNA. This prevented them from doubling their DNA ­ a necessary step for cell division ­ and responding to stress caused by chemotherapy. Harada et al. also noticed that cancer cells lacking MPI did not all react to mannose treatment in exactly the same way. Therefore, future work will address these diverse reactions, potentially providing an opportunity to use the mannose pathway to search for new cancer treatments.

Tight association of CpcL with photosystem I in Anabaena sp. PCC 7120 grown under iron-deficient conditions.

Phycobilisomes (PBSs), which are huge pigment-protein complexes displaying distinctive color variations, bind to photosystem cores for excitation-energy transfer. It is known that isolation of supercomplexes consisting of PBSs and photosystem I (PSI) or PBSs and photosystem II is challenging due to weak interactions between PBSs and the photosystem cores. In this study, we succeeded in purifying PSI-monomer-PBS and PSI-dimer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 grown under iron-deficient conditions by anion-exchange chromatography, followed by trehalose density gradient centrifugation. The absorption spectra of the two types of supercomplexes showed apparent bands originating from PBSs, and their fluorescence-emission spectra exhibited characteristic peaks of PBSs. Two-dimensional blue-native (BN)/SDS-PAGE of the two samples showed a band of CpcL, which is a linker protein of PBS, in addition to PsaA/B. Since interactions of PBSs with PSI are easily dissociated during BN-PAGE using thylakoids from this cyanobacterium grown under iron-replete conditions, it is suggested that iron deficiency for Anabaena induces tight association of CpcL with PSI, resulting in the formation of PSI-monomer-PBS and PSI-dimer-PBS supercomplexes. Based on these findings, we discuss interactions of PBSs with PSI in Anabaena.

Comparative proteomic analysis of glomerular proteins in IgA nephropathy and IgA vasculitis with nephritis.

BACKGROUND: IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN. METHODS: We used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed. RESULTS: More than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-ß inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings. CONCLUSIONS: The present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

Lysine long-chain fatty acylation regulates the TEAD transcription factor.

TEAD transcription factors are responsible for the transcriptional output of Hippo signaling. TEAD activity is primarily regulated by phosphorylation of its coactivators, YAP and TAZ. In addition, cysteine palmitoylation has recently been shown to regulate TEAD activity. Here, we report lysine long-chain fatty acylation as a posttranslational modification of TEADs. Lysine fatty acylation occurs spontaneously via intramolecular transfer of acyl groups from the proximal acylated cysteine residue. Lysine fatty acylation, like cysteine palmitoylation, contributes to the transcriptional activity of TEADs by enhancing the interaction with YAP and TAZ, but it is more stable than cysteine acylation, suggesting that the lysine fatty-acylated TEAD acts as a "stable active form." Significantly, lysine fatty acylation of TEAD increased upon Hippo signaling activation despite a decrease in cysteine acylation. Our results provide insight into the role of fatty-acyl modifications in the regulation of TEAD activity.