Eric Spooner

Reversal of Histone Lysine Trimethylation by the JMJD2 Family of Histone Demethylases

Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases such as LSD1 and JHDM1, which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. We show the JmjC domain-containing protein JMJD2A reversed trimethylated H3-K9/K36 to di- but not mono- or unmethylated products. Overexpression of JMJD2A but not a catalytically inactive mutant reduced H3-K9/K36 trimethylation levels in cultured cells. In contrast, RNAi depletion of the C. elegans JMJD2A homolog resulted in an increase in general H3-K9Me3 and localized H3-K36Me3 levels on meiotic chromosomes and triggered p53-dependent germline apoptosis. Additionally, other human JMJD2 subfamily members also functioned as trimethylation-specific demethylases, converting H3-K9Me3 to H3-K9Me2 and H3-K9Me1, respectively. Our finding that this family of demethylases generates different methylated states at the same lysine residue provides a mechanism for fine-tuning histone methylation.

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily

BACKGROUND Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9

Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9−/− dendritic cells, restored CpG DNA–induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.

Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

“Haploid Human” Genetic screens can provide direct insight into biological processes that are poorly understood. Carette et al. (p. 1231) describe genetic screens using large-scale gene disruption in human cells haploid for all chromosomes except for chromosome 8. One screen was used to identify host factors essential for the activity of cytolethal distending toxin, a toxin found in several pathogenic bacteria. Another screen identified host gene products essential for infection with influenza, and an additional screen revealed genes required for the action of adenosine 5′-diphosphate (ADP)–ribosylating bacterial toxins. This loss-of-function genetic approach in mammalian cells will be widely applicable to study a variety of biological processes and cellular functions. A method identifies human factors required for successful microbial pathogenesis. Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins

Membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum are discharged into the cytosol and degraded by the proteasome. Many of the mammalian components involved in this process remain to be identified. We performed a biochemical search for proteins that interact with SEL1L, a protein that is part of the mammalian HRD1 ligase complex and involved in substrate recognition. SEL1L is crucial for dislocation of Class I major histocompatibility complex heavy chains by the human cytomegalovirus US11 protein. We identified AUP1, UBXD8, UBC6e, and OS9 as functionally important components of this degradation complex in mammalian cells, as confirmed by mutagenesis and dominant negative versions of these proteins.

The Sestrins Interact with GATOR2 to Negatively Regulate the Amino-Acid-Sensing Pathway Upstream of mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway.

Identification and characterization of CTRP9, a novel secreted glycoprotein, from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin

Adiponectin is a major insulin‐sensitizing, multimeric hormone derived from adipose tissue that acts on muscle and liver to regulate whole‐body glucose and lipid metabolism. Here, we describe a novel and highly conserved paralog of adiponectin designated as Clq/TNF‐related protein (CTRP) 9. Of all the CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and females expresses higher levels of the transcript than males. Moreover, its expression levels in ob/ob mice changed in an age‐dependent manner, with significant up‐regulation in younger mice. CTRP9 is a secreted glycoprotein with multiple post‐translational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It is se‐creted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum with levels varying according to sex and metabolic state of mice. Furthermore, CTRP9 and adiponectin can be secreted as heterooligomers when cotrans‐fected into mammalian cells, and in vivo, adiponectin/ CTRP9 complexes can be reciprocally coimmuno‐precipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N‐terminal cysteines or their collagen domains. Furthermore, we show that adiponectin and CTRP9 form heterotrimers. In cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. Adenovirus‐mediated over‐expression of CTRP9 in obese (ob/ob) mice signifi‐cantly lowered serum glucose levels. Collectively, these results suggest that CTRP9 is a novel adipokine, and further study of CTRP9 will yield novel mechanistic insights into its physiological and metabolic function.— Wong, G. W., Krawczyk, S. A., Kitidis‐Mitrokostas, C., Ge, G., Spooner, E., Hug, C., Gimeno, R., Lodish, H. F. Identification and characterization of CTRP9, a novel secreted glycoprotein from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin. FASEB J. 23, 241‐258 (2009)

Signal peptide peptidase is required for dislocation from the endoplasmic reticulum

Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.

Chlamydia trachomatis‐derived deubiquitinating enzymes in mammalian cells during infection

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin‐like proteases (Ulp). The C. trachomatis DUBs react with activity‐based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.

Sortase-catalyzed transformations that improve the properties of cytokines

Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.