Intracellular delivery of Parkin rescues neurons from accumulation of damaged mitochondria and pathological α-synuclein.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by mitochondrial dysfunction, Lewy body formation, and loss of dopaminergic neurons. Parkin, an E3 ubiquitin ligase, is thought to inhibit PD progression by removing damaged mitochondria and suppressing the accumulation of α-synuclein and other protein aggregates. The present study describes a protein-based therapy for PD enabled by the development of a cell-permeable Parkin protein (iCP-Parkin) with enhanced solubility and optimized intracellular delivery.

Cell-permeable parkin proteins suppress Parkinson disease-associated phenotypes in cultured cells and animals.

Parkinson's disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent.

The effect of intracellular protein delivery on the anti-tumor activity of recombinant human endostatin.

Endostatin (ES), a 20 kDa protein derived from the carboxy-terminus of collagen XVIII is a potent angiogenesis inhibitor, but clinical development has been hindered by poor clinical efficacy and insufficient functional information from which to design agents with improved activity. The present study investigated protein uptake by cells as a determinant of ES activity. We developed a cell-permeable ES protein (HM73ES) with enhanced capacity to enter cells by adding a macromolecule transduction domain (MTD).

Antitumor activity of cell-permeable RUNX3 protein in gastric cancer cells.

Purpose: Gastric cancer is a leading cause of cancer death worldwide. Limited therapeutic options highlight the need to understand the molecular changes responsible for the disease and to develop therapies based on this understanding. The goal of this study was to develop cell-permeable (CP-) forms of the RUNT-related transcription factor 3, RUNX3-a candidate tumor suppressor implicated in gastric and other epithelial cancers-to study the therapeutic potential of RUNX3 in the treatment of gastric cancer.

Antitumor activity of cell-permeable p18(INK4c) with enhanced membrane and tissue penetration.

Practical methods to deliver proteins systemically in animals have been hampered by poor tissue penetration and inefficient cytoplasmic localization of internalized proteins. We therefore pursued the development of improved macromolecule transduction domains (MTDs) and tested their ability to deliver therapeutically active p18(INK4c). MTD103 was identified from a screen of 1,500 signal peptides; tested for the ability to promote protein uptake by cells and tissues; and analyzed with regard to the mechanism of protein uptake and the delivery of biologically active p18(INK4c) into cancer cells.

Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-induced cytokine expression in T lymphocytes.

The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the "death" adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1.

Cell-permeable NM23 blocks the maintenance and progression of established pulmonary metastasis.

Occult metastases are a major cause of cancer mortality, even among patients undergoing curative resection. Therefore, practical strategies to target the growth and persistence of already established metastases would provide an important advance in cancer treatment. Here, we assessed the potential of protein therapy using a cell permeable NM23-H1 metastasis suppressor protein.

Observational fear learning involves affective pain system and Cav1.2 Ca2+ channels in ACC.

Fear can be acquired vicariously through social observation of others suffering from aversive stimuli. We found that mice (observers) developed freezing behavior by observing other mice (demonstrators) receive repetitive foot shocks. Observers had higher fear responses when demonstrators were socially related to themselves, such as siblings or mating partners.

Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic.

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic.

Subunit 1 of the prefoldin chaperone complex is required for lymphocyte development and function.

Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function.

Genome maintenance and mutagenesis in embryonic stem cells.

Widespread loss of heterozygosity (LOH) in cancer cells is often thought to result from chromosomal instability caused by mutations affecting DNA repair/genome maintenance; however, the origin of LOH in most tumors is unknown. In a recent study, we examined the ability of carcinogenic agents to induce LOH in diploid mouse embryo-derived stem (ES) cells. Brief exposures to nontoxic levels of several carcinogens stimulated genome-wide LOH, with maximum per-gene frequencies approaching one percent.

Mutagenesis of diploid mammalian genes by gene entrapment.

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector-cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice.

Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability.

Widespread losses of heterozygosity (LOH) in human cancer have been thought to result from chromosomal instability caused by mutations affecting DNA repair/genome maintenance. However, the origin of LOH in most tumors is unknown. The present study examined the ability of carcinogenic agents to induce LOH at 53 sites throughout the genome of normal diploid mouse ES cells.

Cellular genetics of host susceptibility and resistance to virus infection.

Viruses are obligate intracellular parasites that rely upon the host cell for activities essential to their life cycles. Gene-trap mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus replication. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus life cycle and may provide targets for novel antiviral therapies.

Hypomorphic mutation in hnRNP U results in post-implantation lethality.

The present study characterized an embryonic lethal mutation induced by insertion of the U3Neo gene trap retrovirus into an intron of the gene encoding heterogeneous ribonuclear protein U (Hnrnpu), which maps to the distal arm of mouse chromosome 1. Murine hnRNP U was found to be identical to the human protein at all but one of 341 amino acid residues. Embryos homozygous for the provirus showed obvious abnormalities after 6.

Post-entrapment genome engineering: first exon size does not affect the expression of fusion transcripts generated by gene entrapment.

Gene trap mutagenesis in mouse embryonic stem cells has been widely used for genome-wide studies of mammalian gene function. However, while large numbers of genes can be disrupted, individual mutations may suffer from limitations due to the structure and/or placement of targeting vector. To extend the utility of gene trap mutagenesis, replaceable 3' [or poly(A)] gene trap vectors were developed that permit sequences inserted in individual entrapment clones to be engineered by Cre-mediated recombination.

P2P-R expression is genetically coregulated with components of the translation machinery and with PUM2, a translational repressor that associates with the P2P-R mRNA.

P2P-R is a nuclear protein with potential functional roles in the control of gene expression and mitosis. The P2P-R protein also interacts with the p53 and Rb1 tumor suppressor proteins. To search for additional functional associations of P2P-R, we employed the WebQTL database that contains the results of cDNA microarray analysis on forebrain, cerebellum, and hematopoietic stem cell (HSC) specimens of multiple BXD recombinant inbred strains of mice.

Discovery of mammalian genes that participate in virus infection.

Background: Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.

Results: Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment.

Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells.

Background: Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.

Results: In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes.

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection.

Background: Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.

Results: A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis.

Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e.

The high-mobility-group box protein SSRP1/T160 is essential for cell viability in day 3.5 mouse embryos.

The high-mobility-group (HMG) SSRP1 protein is a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, basal and regulated transcription, and DNA repair. To assist in the functional analysis of SSRP1, the Ssrp1 gene was targeted in murine embryonic stem cells, and the mutation was introduced into the germ line. Embryos homozygous for the targeted allele die soon after implantation, and preimplantation blastocysts are defective for cell outgrowth and/or survival in vitro.

Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins.

Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population.

Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.

Background: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.

Protein arginine methyltransferase I: substrate specificity and role in hnRNP assembly.

Prmt1, the major protein arginine methyltransferase in mammalian cells, has been implicated in signal transduction, transcriptional control, and protein trafficking. In the present study, mouse embryonic stem cells homozygous for an essentially null mutation in the Prmt1 gene were used to examine Prmt1 activity and substrate specificity, which by several criteria appeared to be highly specific. First, other methyltransferases did not substitute for the loss of Prmt1 activity.