Bryan E. Snow

Molecular characterization of mitochondrial apoptosis-inducing factor

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.

GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein α subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3′ end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3′ end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class α subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3′ exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function

Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.

Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel.

γ-Aminobutyric acid (GABA)B receptors couple to G o to inhibit N-type calcium channels in embryonic chick dorsal root ganglion neurons. The voltage-independent inhibition, mediated by means of a tyrosine-kinase pathway, is transient and lasts up to 100 seconds. Inhibition of endogenous RGS12, a member of the family of regulators of G-protein signalling, selectively alters the time course of voltage-independent inhibition. The RGS12 protein, in addition to the RGS domain, contains PDZ and PTB domains. Fusion proteins containing the PTB domain of RGS12 alter the rate of termination of the GABAB signal, whereas the PDZ or RGS domains of RGS12 have no observable effects. Using primary dorsal root ganglion neurons in culture, here we show an endogenous agonist-induced tyrosine-kinase-dependent complex of RGS12 and the calcium channel. These results indicate that RGS12 is a multifunctional protein capable of direct interactions through its PTB domain with the tyrosine-phosphorylated calcium channel. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways.

Chk2 regulates irradiation-induced, p53-mediated apoptosis in Drosophila

The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. In Drosophila melanogaster, however, the regulation modes of p53 are still unknown. Overexpression of D. melanogaster p53 (Dmp53) in the eye induced apoptosis, resulting in a small eye phenotype. This phenotype was markedly enhanced by coexpression with D. melanogaster Chk2 (DmChk2) and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of DmChk2. DN DmChk2 also inhibited Dmp53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant had no effect on Dmp53-induced phenotypes. DmChk2 also activated the Dmp53 transactivation activity in cultured cells. Mutagenesis of Dmp53 amino terminal Ser residues revealed that Ser-4 is critical for its responsiveness toward DmChk2. DmChk2 activates the apoptotic activity of Dmp53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating Dmp53. Chk2 may be the ancestral regulator of p53 function.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Significance Current genomic and biochemical analysis revealed mutations in isocitrate dehydrogenase (IDH) genes associated with several neoplasms and a novel enzymatic activity of IDH mutations to catalyze α-ketoglutarate to d-2-hydroxyglutarate, contributing to tumorigenesis. We identified a broad range of IDH1 mutations, including a previously unidentified IDH1-R132Q mutation, in cartilage tumors. Cartilage-specific Col2a1-Cre/ERT2;Idh1-R132 mutant knock-in mice developed multiple enchondroma-like lesions. These data show that mutant Idh in growth-plate cells causes persistence of chondrocytes, giving rise to enchondromas adjacent to the growth cartilage in bone. Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene.

Regulators of G-protein signaling (RGS) constitute a family of GTPase-activating proteins with varying tissue-specific expression patterns and G-protein alpha subunit specificities. Here, we describe the molecular cloning of the human RGS-r/RGS16 cDNA, encoding a predicted polypeptide of 23kDa that shows 86% identity to mouse RGS-r. Northern blot analysis shows that, like the mouse Rgs-r message, hRGS-r mRNA is abundantly expressed in retina, with lower levels of expression in most other tissues examined. Characterization of the genomic organization of the hRGS-r gene shows that it consists of five exons and four introns. We have also mapped the human RGS-r /RGS16 gene to chromosome 1q25-1q31 by fluorescence in situ hybridzation. Analysis of human ESTs reveals that at least five members of the RGS gene family map to chromosome 1q, suggesting that at least part of the RGS family arose through gene duplication. The chromosomal location, retinal abundance, and presumed function of the human RGS-r protein in desensitizing photoreceptor signaling make the RGS-r/RGS16 locus a candidate for mutations responsible for retinitis pigmentosa with para-arteriolar preservation of retinal pigment epithelium (RP-PPRE or RP12), an autosomal recessive disorder previously mapped to 1q31.

Toso controls encephalitogenic immune responses by dendritic cells and regulatory T cells

Significance More than 1 million people worldwide suffer from the debilitating neurological disorder multiple sclerosis (MS). The initiation of MS is associated with sustained inflammation and an autoimmune T-cell response targeting the central nervous system. The activities of Th1 and Th17 effector T cells, which are the main pathogenic drivers of MS, are balanced by regulatory T cells, which dampen inflammation and mitigate disease. Our study describes an important role for the surface receptor Toso in balancing these T-cell subsets and controlling inflammation. Using an animal model of MS, we have developed a preclinical treatment strategy in which Toso-Fc fusion protein ameliorates the inflammatory symptoms of experimental autoimmune encephalomyelitis, an MS-like disease. The ability to mount a strong immune response against pathogens is crucial for mammalian survival. However, excessive and uncontrolled immune reactions can lead to autoimmunity. Unraveling how the reactive versus tolerogenic state is controlled might point toward novel therapeutic strategies to treat autoimmune diseases. The surface receptor Toso/Faim3 has been linked to apoptosis, IgM binding, and innate immune responses. In this study, we used Toso-deficient mice to investigate the importance of Toso in tolerance and autoimmunity. We found that Toso−/− mice do not develop severe experimental autoimmune encephalomyelitis (EAE), a mouse model for the human disease multiple sclerosis. Toso−/− dendritic cells were less sensitive to Toll-like receptor stimulation and induced significantly lower levels of disease-associated inflammatory T-cell responses. Consistent with this observation, the transfer of Toso−/− dendritic cells did not induce autoimmune diabetes, indicating their tolerogenic potential. In Toso−/− mice subjected to EAE induction, we found increased numbers of regulatory T cells and decreased encephalitogenic cellular infiltrates in the brain. Finally, inhibition of Toso activity in vivo at either an early or late stage of EAE induction prevented further disease progression. Taken together, our data identify Toso as a unique regulator of inflammatory autoimmune responses and an attractive target for therapeutic intervention.