Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short ß-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.

Use of Mitotic Protein Kinase Inhibitors and Phospho-Specific Antibodies to Monitor Protein Phosphorylation During the Cell Cycle.

Reversible protein phosphorylation regulates the transitions between different phases of the cell cycle ensuring proper segregation of the duplicated genome into two daughter cells. Protein kinases and protein phosphatases establish the appropriate phosphorylation stoichiometries in diverse substrates maintaining genomic stability as a cell undergoes this complex process. Along with regulating common substrates, these opposing enzymes regulate one another by fine-tuning each other's activity both spatially and temporally throughout mitosis. Protein phosphatase catalytic subunits work together with regulatory proteins, which control their localization, activity, and specificity. Protein phosphatase 1 (PP1) recognizes its regulatory proteins via a short linear interaction motif (SLIM) called the "RVxF" motif. A subset of proteins with these "RVxF" motifs contain a phosphorylatable amino acid (S/T) at the 'x' position.Here, we describe methods to generate, affinity purify and utilize phospho-specific antibodies to monitor phosphorylation sites during the cell cycle and the appropriate use of mitotic kinase inhibitors. More specifically, we employ phospho-specific antibodies, which recognize phosphorylated RVp[S/T]F motif-containing proteins, to monitor the phosphorylation status of these motifs throughout the cell cycle. Furthermore, we use mitotic kinase inhibitors to examine the effect of kinase inhibition on the phosphorylation status of multiple RV[S/T]F motifs using these phospho-specific antibodies.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

Hypermethylation of Auxin-Responsive Motifs in the Promoters of the Transcription Factor Genes Accompanies the Somatic Embryogenesis Induction in Arabidopsis.

The auxin-induced embryogenic reprogramming of plant somatic cells is associated with extensive modulation of the gene expression in which epigenetic modifications, including DNA methylation, seem to play a crucial role. However, the function of DNA methylation, including the role of auxin in epigenetic regulation of the SE-controlling genes, remains poorly understood. Hence, in the present study, we analysed the expression and methylation of the TF genes that play a critical regulatory role during SE induction (LEC1, LEC2, BBM, WUS and AGL15) in auxin-treated explants of Arabidopsis. The results showed that auxin treatment substantially affected both the expression and methylation patterns of the SE-involved TF genes in a concentration-dependent manner. The auxin treatment differentially modulated the methylation of the promoter (P) and gene body (GB) sequences of the SE-involved genes. Relevantly, the SE-effective auxin treatment (5.0 µM of 2,4-D) was associated with the stable hypermethylation of the P regions of the SE-involved genes and a significantly higher methylation of the P than the GB fragments was a characteristic feature of the embryogenic culture. The presence of auxin-responsive (AuxRE) motifs in the hypermethylated P regions suggests that auxin might substantially contribute to the DNA methylation-mediated control of the SE-involved genes.

Regulation of Small GTPase Prenylation in the Nervous System.

Mevalonate pathway inhibitors have been extensively studied for their roles in cholesterol depletion and for inhibiting the prenylation and activation of various proteins. Inhibition of protein prenylation has potential therapeutic uses against neurological disorders, like neural cancers, neurodegeneration, and neurotramatic lesions. Protection against neurodegeneration and promotion of neuronal regeneration is regulated in large part by Ras superfamily small guanosine triphosphatases (GTPases), particularly the Ras, Rho, and Rab subfamilies. These proteins are prenylated to target them to cellular membranes. Prenylation can be specifically inhibited through altering the function of enzymes of the mevalonate pathway necessary for isoprenoid production and attachment to target proteins to elicit a variety of effects on neural cells. However, this approach does not address how prenylation affects a specific protein. This review focuses on the regulation of small GTPase prenylation, the different techniques to inhibit prenylation, and how this inhibition has affected neural cell processes.

A suppressive oligodeoxynucleotide expressing TTAGGG motifs modulates cellular energetics through the mTOR signaling pathway.

Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were dose- and time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.

A Disintegrin and Metalloproteinase (ADAM) and ADAM with thrombospondin motifs (ADAMTS) family in vascular biology and disease.

A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.

In Vitro Characterization of a Potent p53-MDM2 Inhibitor, RG7112 in Neuroblastoma Cancer Cell Lines.

Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality and morbidity of the disease, particularly in relapsed patients. The tumor suppressor protein p53 plays a critical role in multiple signaling pathways to maintain cellular hemostasis. Dysregulation of p53 protein and/or molecular aberrations have been associated with multiple human malignancies. p53 stability and protein activity is negatively regulated by the E3 ubiquitin ligase (MDM2). Thus, targeting p53-MDM2 protein-protein interaction is a feasible and promising therapeutic strategy to restore the physiological function of p53 in cancer cells. RG7112 is a highly potent and selective small molecule inhibitor, which target a unique structure located within p53 binding motif of MDM2. Methods: The efficacy of RG7112 in vitro using NB cell lines was examined. Two wild-type (WT)-p53 NB cell lines IMR5 and LAN-5, a mutant p53 cell line SK-N-BE(2), and a WT-p53/p14 deleted cell line SH-EP were employed. Results: Data showed that RG7112 significantly reduced cellular viability of IMR5 (IC50, 562 nM) and LAN-5 (IC50, 430 nM), but not SK-N-BE(2) and SH-EP cells. Further, RG7112 restores p53 and p21 protein levels in IMR5 and LAN-5 in a dose-dependent manner. RG7112 induces cell cycle arresting (60% G1 arresting) in WT-p53 cells (IMR5), but no pronounced effect observed in SK-N-BE(2). In this study, 15 different drugs in combination with RG7112 in IMR5 cell line and identified venetoclax (Bcl-2/Bcl-xL inhibitor) as a promising candidate were evaluated. Conclusions: Taken together, these findings provide initial proof-of-concept data for further investigations of RG7112 in selected subgroups of NB patients.

Protein targeting chimeric molecules specific for bromodomain and extra-terminal motif family proteins are active against pre-clinical models of multiple myeloma.

Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.

Interference of norepinephrine transporter trafficking motif attenuates amphetamine-induced locomotor hyperactivity and conditioned place preference.

Amphetamine (AMPH)-mediated norepinephrine transporter (NET) downregulation requires NET-T258/S259 trafficking motif. The present study utilizes cell permeable NET-T258/S259 motif interfering peptide, which blocks AMPH-induced NET downregulation, to explore the role of this form of NET regulation in AMPH-mediated behaviors. In rats receiving intra-accumbal microinjections of TAT-conjugated peptides encompassing NET-T258/S259 motif, acute systemic AMPH failed to inhibit NE transport in the TAT-NET-T258/S259 wild-type (WT) peptide injected hemisphere but not in the vehicle or scrambled peptide injected hemisphere. Acute AMPH-induced hyperactivity was significantly reduced in rats receiving intra-accumbal TAT-NET-T258/S259 WT peptide compared to those receiving intra-accumbal vehicle or TAT-NET-T258A/S259A mutant peptide or corresponding TAT-conjugated scrambled peptide. Basal locomotor activity was not altered by peptide infusions alone. Similarly AMPH-induced locomotor sensitization was significantly reduced in rats receiving intra-accumbal TAT-NET-T258/S259 WT peptide prior to AMPH challenge and not in rats receiving the mutant or scrambled peptide. In conditioned place preference (CPP) paradigm, a single bilateral intra-accumbal microinjection of TAT-NET-T258/S259 WT peptide prior to CPP testing significantly reduced AMPH-induced CPP expression. Likewise, a single bilateral intra-accumbal microinjection of TAT-NET-T258/S259 WT peptide prior to drug-challenge significantly attenuated AMPH-primed CPP reinstatement. On the other hand, bilateral intra-accumbal microinjection of scrambled peptide did not affect AMPH-induced CPP expression or reinstatement. These data demonstrate a role for T258/S259-dependent NET regulation in AMPH-induced hyperactivity and sensitization as well as AMPH-induced CPP expression and reinstatement.

Central Role of the Copper-Binding Motif in the Complex Mechanism of Action of Ixosin: Enhancing Oxidative Damage and Promoting Synergy with Ixosin B.

Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the amino-terminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen- and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.

The closed conformation of the LDL receptor is destabilized by the low Ca(++) concentration but favored by the high Mg(++) concentration in the endosome.

The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closed conformation important for recycling, by interaction of two modules of the ligand binding domain (LR4-5) and a ß-propeller motif. Here, we investigate by SPR the interactions between those two modules and the ß-propeller. Our results indicate that the two modules cooperate to bind the ß-propeller. The binding is favored by low pH and by high [Ca(++)]. Our data show that Mg(++), at high concentration in the endosome, favors the formation of the closed conformation by replacing the structuring effect of Ca(++) in LR5. We propose a sequential model of LDL release where formation of the close conformation follows LDL release.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

Sequence-motif detection of NAD(P)-binding proteins: discovery of a unique antibacterial drug target.

Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

Acetylation of the KXGS motifs in tau is a critical determinant in modulation of tau aggregation and clearance.

The accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of tauopathies, including Alzheimer's disease (AD) and chronic traumatic encephalopathy, but effective therapies directly targeting the tau protein are currently lacking. Herein, we describe a novel mechanism in which the acetylation of tau on KXGS motifs inhibits phosphorylation on this same motif, and also prevents tau aggregation. Using a site-specific antibody to detect acetylation of KXGS motifs, we demonstrate that these sites are hypoacetylated in patients with AD, as well as a mouse model of tauopathy, suggesting that loss of acetylation on KXGS motifs renders tau vulnerable to pathogenic insults. Furthermore, we identify histone deacetylase 6 (HDAC6) as the enzyme responsible for the deacetylation of these residues, and provide proof of concept that acute treatment with a selective and blood-brain barrier-permeable HDAC6 inhibitor enhances acetylation and decreases phosphorylation on tau's KXGS motifs in vivo. As such, we have uncovered a novel therapeutic pathway that can be manipulated to block the formation of pathogenic tau species in disease.

Prevelance of common YMDD motif mutations in long term treated chronic HBV infections in a Turkish population.

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/ RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/ or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.

In silico cross seeding of Aß and amylin fibril-like oligomers.

Recent epidemiological data have shown that patients suffering from Type 2 Diabetes Mellitus have an increased risk to develop Alzheimer's disease and vice versa. A possible explanation is the cross-sequence interaction between Aß and amylin. Because the resulting amyloid oligomers are difficult to probe in experiments, we investigate stability and conformational changes of Aß-amylin heteroassemblies through molecular dynamics simulations. We find that Aß is a good template for the growth of amylin and vice versa. We see water molecules permeate the ß-strand-turn-ß-strand motif pore of the oligomers, supporting a commonly accepted mechanism for toxicity of ß-rich amyloid oligomers. Aiming for a better understanding of the physical mechanisms of cross-seeding and cell toxicity of amylin and Aß aggregates, our simulations also allow us to identify targets for the rational design of inhibitors against toxic fibril-like oligomers of Aß and amylin oligomers.

Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors.

Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.

Ca(2+) binding to the ExDxD motif regulates the DNA cleavage specificity of a promiscuous endonuclease.

Most of the restriction endonucleases (REases) are dependent on Mg(2+) for DNA cleavage, and in general, Ca(2+) inhibits their activity. R.KpnI, an HNH active site containing ßßα-Me finger nuclease, is an exception. In presence of Ca(2+), the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg(2+)-induced promiscuous activity. To elucidate the mechanism of unusual Ca(2+)-mediated activity, we generated alanine variants in the putative Ca(2+) binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca(2+). We demonstrate that ExDxD residues are involved in Ca(2+) coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca(2+)-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca(2+) binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg(2+), the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca(2+) binding motif that confers site specific cleavage upon Ca(2+) binding is also critical for the promiscuous activity of the Mg(2+)-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

YMDD motif mutations in chronic hepatitis B antiviral treatment naïve patients: a multi-center study.

OBJECTIVE: This study aimed to determine the natural prevalence of variants of tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels. METHODS: A total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA. RESULTS: YMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level. CONCLUSION: Mutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.