ABSTRACT
The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.
Subject(s)
BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endonucleases/metabolism , Multifunctional Enzymes/metabolism , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Cell Line , DNA/metabolism , DNA Damage , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Endonucleases/genetics , Genes, BRCA1/physiology , Humans , Multifunctional Enzymes/genetics , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolismABSTRACT
Purinergic receptor P2X3 has been linked to analgesia in a number of pre-clinical models of pain, and is expressed in the human pain perception pathway. Only few P2X3-selective antagonists have been reported to date. This Letter describes the SAR and in vivo analgesic profile of a novel scaffold of selective P2X3 antagonists.
Subject(s)
Analgesics/chemical synthesis , Chronic Pain/drug therapy , Purinergic P2 Receptor Antagonists/chemical synthesis , Pyrimidinones/chemical synthesis , Pyrroles/chemical synthesis , Receptors, Purinergic P2X3/chemistry , Analgesics/administration & dosage , Analgesics/therapeutic use , Animals , Chronic Pain/metabolism , Dose-Response Relationship, Drug , High-Throughput Screening Assays , Humans , Injections, Spinal , Injections, Subcutaneous , Purinergic P2 Receptor Antagonists/administration & dosage , Purinergic P2 Receptor Antagonists/therapeutic use , Pyrimidinones/administration & dosage , Pyrimidinones/therapeutic use , Pyrroles/administration & dosage , Pyrroles/therapeutic use , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X3/metabolism , Small Molecule LibrariesABSTRACT
Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.
Subject(s)
Enkephalins/metabolism , GTP-Binding Proteins/metabolism , Neurons, Afferent/metabolism , Nociceptors/metabolism , Protein Precursors/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Animals , Brain Chemistry , Calcium/metabolism , Embryo, Mammalian/physiology , Enkephalins/genetics , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Humans , In Situ Hybridization , Ligands , Molecular Sequence Data , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Neurons, Afferent/drug effects , Nociceptors/drug effects , Opioid Peptides/metabolism , Phylogeny , Protein Binding , Protein Precursors/genetics , Rats , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Sequence Alignment , Tissue DistributionABSTRACT
Transient gene expression in human embryo kidney 293 (HEK293) cells is an established approach for the rapid production of large amounts of recombinant proteins (r-proteins). Milligram to gram quantities of r-proteins can be typically obtained within less than 10 days following transfection. In this chapter, we describe a simple and robust transfection process of suspension-growing human embryo kidney 293 cells using two commercially available serum-free media and polyethylenimine as the transfection reagent. This chapter provides examples for the production and purification of a his-tagged recombinant protein and two monoclonal antibodies.
Subject(s)
Transfection/methods , Culture Media, Serum-Free , HEK293 Cells , Humans , Polyethyleneimine/chemistry , Recombinant Proteins/geneticsABSTRACT
For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.
Subject(s)
Epstein-Barr Virus Nuclear Antigens/genetics , Polyethyleneimine , Transfection/methods , Trastuzumab/biosynthesis , Animals , CHO Cells , Cell Count , Cricetulus , Gene ExpressionABSTRACT
1. Human formyl peptide-receptor-like-1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL-1 as a high-affinity beta-chemokine receptor for an N-terminally truncated form of the CKbeta8 (CCL23/MPIF-1) splice variant CKbeta8-1 (22-137 aa). 2. RT-PCR analysis of mRNA derived from human tissues and cells revealed a predominant expression of FPRL-1 in inflammatory cells, particularly in neutrophils. 3. Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293s) cells coexpressing FPRL-1 and Galpha(16), demonstrated FPRL-1 is a functional high-affinity receptor for CKbeta8-1 (46-137 aa, sCKbeta8-1), with pEC(50) values of 9.13 and 8.85, respectively. 4. The FPRL-1 activation in CHO-K1 cells is mediated by Galpha(i)/Galpha(o) proteins, as assessed by pertussis toxin sensitivity and inhibition of forskolin-induced cyclic AMP accumulation. 5. Binding experiments were performed with a radio-iodinated synthetic peptide, [(125-)I]-WKYMVm, a known potent FPRL-1 agonist. CHO-K1 cell membranes expressing FPRL-1 bound [(125-)I]-WKYMVm with a K(d) value of 9.34. Many known FPRL-1 agonists were tested and sCKbeta8-1 was the most effective nonsynthetic ligand in displacing the radiolabelled agonist, with a pIC(50) of 7.97. 6. The functional significance of sCKbeta8-1 interaction with FPRL-1 was further demonstrated by the activation of polymorphonuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL-1 by specific blockade of PMNs activation in the presence of an FPRL-1 antibody.
Subject(s)
Chemokines, CC/chemistry , Chemokines, CC/pharmacology , Receptors, Formyl Peptide/drug effects , Receptors, Lipoxin/drug effects , Amino Acid Sequence , Animals , CHO Cells , Calcium/metabolism , Cell Movement/drug effects , Chemokines, CC/metabolism , Chemotaxis/drug effects , Cricetinae , Drug Evaluation, Preclinical/methods , Female , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Gene Expression , Humans , Iodine Radioisotopes/metabolism , Kidney/cytology , Kidney/drug effects , Neutrophils/drug effects , Neutrophils/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Receptors, Formyl Peptide/genetics , Receptors, Formyl Peptide/metabolism , Receptors, G-Protein-Coupled , Receptors, Lipoxin/genetics , Receptors, Lipoxin/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The sensory neuron-specific G protein coupled receptors (SNSRs) have been described as a family of receptors whose expression in small diameter sensory neurons in the trigeminal and dorsal root ganglia suggests an implication in nociception. To date, the physiological function(s) of SNSRs remain unknown. Hence, the aim of the present study was to determine the effects of rat SNSR1 activation on nociception in rats. The pharmacological characterization of rat SNSR1 was initially performed in vitro to identify a specific ligand, which could be used subsequently in the rat for physiological testing. Among all ligands tested, gamma2-MSH was the most potent at activating rat SNSR1. Structure-activity relationship studies revealed that the active moiety recognized by rat SNSR1 was the C-terminal part of gamma2-MSH. The radiolabeled C-terminal part of gamma2-MSH, gamma2-MSH-6-12, bound with high affinity to membranes derived from rat skin and spinal cord, demonstrating the presence of receptor protein at both the proximal and distal terminals of dorsal root ganglia. To investigate the physiological role of SNSR, specific ligands to rat SNSR1 were tested in behavioral assays of pain sensitivity in rats. Selective rat SNSR1 agonists produced spontaneous pain behavior, enhanced heat and mechanical sensitivity when injected intradermally, and heat hypersensitivity when injected centrally, consistent with the localization of rat SNSR1 protein at central and peripheral sites. Together, these results clearly indicate that the SNSR1 plays a role in nociception and may provide novel therapeutic opportunities for analgesia.