Benedikt M. Kessler

Acetylation of the C terminus of Ku70 by CBP and PCAF controls bax-mediated apoptosis

Apoptosis is a key tumor suppression mechanism that can be initiated by activation of the proapoptotic factor Bax. The Ku70 DNA end-joining protein has recently been shown to suppress apoptosis by sequestering Bax from mitochondria. The mechanism by which Bax is regulated remains unknown. Here, we identify eight lysines in Ku70 that are targets for acetylation in vivo. Five of these, K539, K542, K544, K533, and K556, lie in the C-terminal linker domain of Ku70 adjacent to the Bax interaction domain. We show that CBP and PCAF efficiently acetylate K542 in vitro and associate with Ku70 in vivo. Mimicking acetylation of K539 or K542 or treating cells with deacetylase inhibitors abolishes the ability of Ku70 to suppress Bax-mediated apoptosis. We demonstrate that increased acetylation of Ku70 disrupts the Ku70-Bax interaction and coincides with cytoplasmic accumulation of CBP. These results shed light on the role of acetyltransferases as tumor suppressors.

Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.

The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

A C‐terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site‐directed probe that irreversibly modifies a subset of Ub C‐terminal hydrolases (UCHs) and Ub‐specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [125I]UbVS labeling with N‐ethylmaleimide and Ub aldehyde, but also by genetic analysis. [125I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [125I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S‐associated USP14 with [125I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Summary The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

Arginine methylation regulates the p53 response

Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity of p53. Furthermore, PRMT5 depletion triggers p53-dependent apoptosis. Thus, methylation on arginine residues is an underlying mechanism of control during the p53 response.

Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Summary Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

Neuronal loss and brain atrophy in mice lacking cathepsins B and L

Cathepsins B and L are widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. However, specific roles remain to be validated in vivo. Here we show that combined deficiency of cathepsins B and L in mice is lethal during the second to fourth week of life. Cathepsin B−/−/L−/− mice reveal a degree of brain atrophy not previously seen in mice. This is because of massive apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive astrocytosis and is preceded by an accumulation of ultrastructurally and biochemically unique lysosomal bodies in large cortical neurons and by axonal enlargements. Our data demonstrate a pivotal role for cathepsins B and L in maintenance of the central nervous system.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing

Modifying the Modifier Covalent modification of proteins provides an important means whereby their function is regulated. Hydroxylation, catalyzed by oxygenase enzymes, plays an important role in the response to hypoxia, for example. The human protein Jmjd6 has been thought to act as an oxygenase, catalyzing the demethylation of histone H3 at arginine-2 and histone H4 at arginine-3. Webby et al. (p. 90) now show that Jmjd6 interacts with the messenger RNA splicing factor U2AF65 and acts to hydroxylate this protein at lysine residues, modifications also seen in vivo. Furthermore, Jmjd6 modulates the alternative splicing of both an endogenous gene and an introduced mini-gene. An oxygenase with an important role in vertebrate development hydroxylates a messenger RNA splicing factor. The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate–dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase. Jmjd6 is shown to change alternative RNA splicing of some, but not all, of the endogenous and reporter genes, supporting a specific role for Jmjd6 in the regulation of RNA splicing.

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7−/− erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119+/CD71High stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.