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1.
bioRxiv ; 2024 Mar 12.
Article in English | MEDLINE | ID: mdl-38559047

ABSTRACT

WWC2 (WW and C2 domain-containing protein) is implicated in several neurological disorders, however its function in the brain has yet to be determined. Here, we demonstrate that WWC2 interacts with inhibitory but not excitatory postsynaptic scaffolds, consistent with prior proteomic identification of WWC2 as a putative component of the inhibitory postsynaptic density. Using mice lacking WWC2 expression in excitatory forebrain neurons, we show that WWC2 suppresses GABA A R incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABA A R recycling to the membrane. Inhibitory synaptic transmission is dysregulated in CA1 pyramidal cells lacking WWC2. Furthermore, unlike the WWC2 homolog KIBRA (WWC1), a key regulator of AMPA receptor trafficking at excitatory synapses, deletion of WWC2 does not affect synaptic AMPAR expression. In contrast, loss of KIBRA does not affect GABA A R membrane expression. These data reveal unique, synapse class-selective functions for WWC proteins as regulators of ionotropic neurotransmitter receptors and provide insight into mechanisms regulating GABA A R membrane expression.

2.
iScience ; 25(12): 105623, 2022 Dec 22.
Article in English | MEDLINE | ID: mdl-36465112

ABSTRACT

A growing body of human literature implicates KIBRA in memory and neurodevelopmental disorders. Memory and the cellular substrates supporting adaptive cognition change across development. Using an inducible KIBRA knockout mouse, we demonstrate that adult-onset deletion of KIBRA in forebrain neurons impairs long-term spatial memory and long-term potentiation (LTP). These LTP deficits correlate with adult-selective decreases in extrasynaptic AMPA receptors under basal conditions, and we identify a role for KIBRA in LTP-induced AMPAR upregulation. In contrast, juvenile-onset deletion of KIBRA in forebrain neurons did not affect LTP and had minimal effects on basal AMPAR expression. LTP did not increase AMPAR protein expression in juvenile WT mice, providing a potential explanation for juvenile resilience to KIBRA deletion. These data suggest that KIBRA serves a unique role in adult hippocampal function through regulation of basal and activity-dependent AMPAR proteostasis that supports synaptic plasticity.

3.
J Oral Maxillofac Surg ; 69(9): 2452-9, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21684654

ABSTRACT

PURPOSE: Nanoparticle drug delivery offers a potential solution in the treatment of cancer. Using a heterotopic tumor model for head and neck squamous cell carcinoma (HNSCC), tumors of variable folate binding protein-alpha (FBP-α) have been treated to delineate receptor necessity as well as efficacy and toxicity of folate targeted chemotherapy. MATERIALS AND METHODS: University of Michigan Squamous Cell Carcinoma (UM-SCC) and American Type Culture Collection (ATCC) cell lines were screened using quantitative real-time polymerase chain reaction for FBP-α expression. Acetylated generation 5 dendrimers conjugated to the targeting moiety folic acid and the therapeutic moiety methotrexate were fabricated and administered to severe combined immunodeficiency (SCID) CB-17 mice inoculated with UM-SCC-1, UM-SCC-17B, and UM-SCC-22B cancer cells. Mice were injected with targeted therapy, free methotrexate, or saline control and monitored for drug efficacy and toxicity. RESULTS: Targeted therapy was effective relative to receptor level expression. Targeted therapy could be delivered in molar doses 3 times that of free drug. The treatment of a high folate expression tumor cell population was noted to have increased efficacy over saline (P < .01) and free methotrexate (P = .03) as well as decreased systemic toxicity. CONCLUSIONS: This report represents the first translation of dendrimer-based chemotherapy to HNSCC and underscores its effectiveness as an antitumor agent in human cancer cell lines with lower levels of FBP-α than the in vitro and in vivo models previously reported.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Head and Neck Neoplasms/drug therapy , Methotrexate/therapeutic use , Molecular Targeted Therapy/methods , Animals , Antineoplastic Agents/toxicity , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Dendrimers , Disease Models, Animal , Female , Folate Receptor 1/biosynthesis , Head and Neck Neoplasms/metabolism , Humans , Methotrexate/toxicity , Mice , Mice, SCID , Neoplasm Transplantation
4.
Bioorg Med Chem Lett ; 20(21): 6250-3, 2010 Nov 01.
Article in English | MEDLINE | ID: mdl-20833544

ABSTRACT

Two morphine prodrugs ('PDA' and 'PDB') were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5-10min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse>rat>guinea pig>human). Morphine's release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.


Subject(s)
Analgesics, Opioid/blood , Analgesics, Opioid/pharmacokinetics , Morphine/blood , Morphine/pharmacokinetics , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Animals , Carboxylic Ester Hydrolases/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Guinea Pigs , Humans , Hydrolysis , Indicators and Reagents , Magnetic Resonance Spectroscopy , Mice , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Opioid, mu/metabolism , Reference Standards
5.
Anticancer Drugs ; 21(2): 186-92, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20010426

ABSTRACT

Our earlier studies have shown the in vitro and in vivo targeting of a generation 5 (G5) dendrimer-based multifunctional conjugate that contained folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. To clinically apply the synthesized G5-FA-MTX nanotherapeutic, it is important that the anticancer conjugate elicits cytotoxicity specifically and consistently. Toward this objective, we evaluated the large-scale synthesis of a G5-FA-MTX conjugate (Lot # 123-34) for its cytotoxic potential and specificity in vitro and in vivo. The cytotoxicity and specificity were tested by using a coculture assay in which FA receptor-expressing and nonexpressing cells (KB and SK-BR-3 cells, respectively) were cultured together and preferential killing was examined. The in-vitro data were compared with the in-vivo data obtained from a heterogeneous xenograft tumor model. The animal model of the artificial heterogeneous xenograft tumor showed that the nanotherapeutic was preferentially cytotoxic to KB cells.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Breast Neoplasms/drug therapy , Cell Proliferation/drug effects , Dendrimers/chemistry , Folic Acid/metabolism , Methotrexate/pharmacology , Nanostructures/chemistry , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Drug Carriers/chemical synthesis , Drug Carriers/chemistry , Drug Delivery Systems , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , KB Cells/drug effects , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays
6.
EMBO J ; 28(12): 1792-802, 2009 Jun 17.
Article in English | MEDLINE | ID: mdl-19461582

ABSTRACT

The accurate segregation of DNA is essential for the faithful inheritance of genetic information. Segregation of the prototypical P1 plasmid par system requires two proteins, ParA and ParB, and a centromere. When bound to ATP, ParA mediates segregation by interacting with centromere-bound ParB, but when bound to ADP, ParA fulfils a different function: DNA-binding transcription autoregulation. The structure of ParA is unknown as is how distinct nucleotides arbitrate its different functions. To address these questions, we carried out structural and biochemical studies. Crystal structures show that ParA consists of an elongated N-terminal alpha-helix, which unexpectedly mediates dimerization, a winged-HTH and a Walker-box containing C-domain. Biochemical data confirm that apoParA forms dimers at physiological concentrations. Comparisons of four apoParA structures reveal a strikingly flexible dimer interface that allows ParA to adopt multiple conformations. The ParA-ADP structure shows that ADP-binding activates DNA binding using a bipartite mechanism. First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA-binding basic motifs that we show are critical for operator binding.


Subject(s)
Adenosine Diphosphate/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Plasmids/metabolism , Transcription, Genetic , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Apoproteins/chemistry , Apoproteins/metabolism , Crystallography, X-Ray , DNA, Bacterial/metabolism , Escherichia coli Proteins/metabolism , Helix-Turn-Helix Motifs , Models, Molecular , Molecular Sequence Data , Pliability , Polymers , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Sequence Homology, Amino Acid
7.
Nature ; 450(7173): 1268-71, 2007 Dec 20.
Article in English | MEDLINE | ID: mdl-18097417

ABSTRACT

The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon-helix-helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Centromere/genetics , Centromere/metabolism , Chromosome Segregation , Plasmids/chemistry , Plasmids/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Crystallography, X-Ray , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Models, Molecular , Molecular Conformation , Plasmids/genetics , Repressor Proteins/genetics , Staphylococcus aureus
8.
Biochemistry ; 44(24): 8757-69, 2005 Jun 21.
Article in English | MEDLINE | ID: mdl-15952782

ABSTRACT

The human neuronal cannabinoid receptor (CB1) is a G-protein-coupled receptor (GPCR) triggered by the psychoactive ingredients in marijuana, as well as endogenous cannabinoids produced in the brain. As with most GPCRs, the mechanism of CB1 activation is poorly understood. In this work, we have assessed the role of cysteine residues in CB1 ligand binding and activation, and demonstrate a method for mapping key determinants in CB1 structure and function. Through mutational analysis, we find that only two cysteines, C257 and C264, are required for high-level expression and receptor function. In addition, through cysteine reactivity studies, we find that a cysteine in transmembrane helix seven, C386 (C7.42), is reactive toward methanethiosulfonate (MTS) sulfhydryl labeling agents, and is thus solvent accessible. Interestingly, steric bulk introduced at this site, either through MTS labeling or by mutation, inhibits binding of the antagonist drug SR141716A (also known as Rimonabant or Accomplia), but does not affect the binding of the agonist CP55940. Our subsequent modeling studies suggest this effect is caused by steric clash of the modified C386 residue with the piperidine ring of SR141716A and/or disruption of an aromatic microdomain in the binding pocket. On the basis of these results, we hypothesize that bound SR141716A inhibits the ability of transmembrane helix 6 to move during formation of the functionally active receptor state.


Subject(s)
Cysteine/analysis , Piperidines/pharmacokinetics , Pyrazoles/pharmacokinetics , Receptors, Cannabinoid/chemistry , Receptors, Cannabinoid/metabolism , Amino Acid Sequence , Binding Sites , Cannabinoid Receptor Antagonists , Databases, Protein , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Rimonabant , Sequence Alignment , Sequence Homology, Amino Acid
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