Pranab K. Chanda

Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1.

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

Regulator of G Protein Signaling Z1 (RGSZ1) Interacts with Gαi Subunits and Regulates Gαi-mediated Cell Signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein α subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Gαz subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Gαz, but also with Gαi, as supported byin vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind35S-labeled Gαi1 protein in anAlF 4 − dependent manner. The interaction between RGSZ1 and Gαi was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Gαi1 in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Gαi-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an α2-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Gαi and regulates Gαi-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.

Snapin interacts with the N-terminus of regulator of G protein signaling 7

The N-terminus of regulator of G protein signaling 7 (RGS7) contains a dishevelled/egl-10/pleckstrin (DEP) domain of unknown function. To gain insight into its function, we used yeast two-hybrid analysis to screen a human whole brain cDNA library in order to identify proteins that interact specifically with the N-terminus of human RGS7 (amino acid residues 1-248). From this analysis, we identified snapin, a protein associated with the SNARE complex in neurons, as an interactor with the N-terminus of RGS7. Deletion mutation analysis in yeast demonstrated that the interaction between RGS7 and snapin is specific and is mediated primarily by amino acid residues 1-69 of RGS7 (which contains the proximal portion of the DEP domain). The interaction between RGS7 and snapin was also demonstrated in mammalian cells by coimmunoprecipitation and pull-down assays. Our results suggest that RGS7 could play a role in synaptic vesicle exocytosis through its interaction with snapin.

Ultrastructural characterization of human immunodeficiency virus type 1 gag-containing particles assembled in a recombinant adenovirus vector system

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with amolecular mass of 18.7 kDa, is a matrix metalloproteinase which has beenassociated with pathologies such as arthritis and cancer. The assignments ofthe 1H, 15N, 13CO and13C resonances, determination of the secondary structure andanalysis of 15N relaxation data of the inhibitor-freecatalytic fragment of recombinant human fibroblast collagenase (MMP-1) arepresented. It is shown that MMP-1 is composed of a β-sheet consistingof five β-strands in a mixed parallel and antiparallel arrangement(residues 13–19, 48–53, 59–65, 82–85 and94–99) and three α-helices (residues 27–43, 112–124and 150–160). This is nearly identical to the secondary structuredetermined from the refined X-ray crystal structures of inhibited MMP-1. Themajor difference observed between the NMR solution structure ofinhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is thedynamics of the active site. The 2D 15N-1H HSQCspectra, the lack of information in the 15N-edited NOESYspectra, and the generalized order parameters (S2) determinedfrom 15N T1, T2 and NOE datasuggest a slow conformational exchange for residues comprising the activesite (helix B, zinc ligated histidines and the nearby loop region) and ahigh mobility for residues Pro138-Gly144 in thevicinity of the active site for inhibitor-free collagenase. In contrast tothe X-ray structures, only the slow conformational exchange is lost in thepresence of an inhibitor.

Heteroaryl and Cycloalkyl Sulfonamide Hydroxamic Acid Inhibitors of Matrix Metalloproteinases

Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.

Simultaneous determination of 2‐arachidonoylglycerol, 1‐arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry

Endocannabinoids (ECs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2-AG. Identification and quantification of 2-AG, 1-AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2-AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2-AG, 1-AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2-AG, 1-AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02-20, 0.01-10 and 0.05-50 ng/mg tissue for 2-AG, 1-AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2-AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2-AG, 1-AG and AA in mouse brain and adipose tissues.

d-Amino Acid Oxidase Activity Is Inhibited by an Interaction with Bassoon Protein at the Presynaptic Active Zone

Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the worlds population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding d-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist d-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic d-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.

A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides.

Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25-40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G+C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.

Validation of a medium-throughput electrophysiological assay for KCNQ2/3 channel enhancers using IonWorks HT.

Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput electrophysiological assay by using the IonWorks platform. Screening of 20 CHO-K1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at —10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at —80 mV and depolarize to —10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC50s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R 2 > 0.9) between the 2 assays, thus demonstrating the highly predictive nature of the IonWorks assay. (Journal of Biomolecular Screening 2007;1059-1067)