Altered cofactor regulation with disease-associated p97/VCP mutations.

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.

Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Identification of a novel role of ZMIZ2 protein in regulating the activity of the Wnt/ß-catenin signaling pathway.

ZMIZ2, also named ZIMP7, is a protein inhibitor of activated STAT (PIAS)-like protein and a transcriptional coactivator. In this study, we investigated the interaction between ZMIZ2 and ß-catenin, a key regulator of the Wnt signaling pathway. We demonstrated that the expression of exogenous ZMIZ2 augments TCF (T cell factor) and ß-catenin-mediated transcription. In contrast, shRNA knockdown of ZMIZ2 expression specifically represses the enhancement of TCF/ß-catenin-mediated transcription by ZMIZ2. Using Wnt3a-conditioned medium, we demonstrated that ZMIZ2 can enhance Wnt ligand-induced TCF/ß-catenin-mediated transcription. We also showed a promotional role of ZMIZ2 in enhancing ß-catenin downstream target gene expression in human cells and in Zmiz2 null (Zmiz2(-/-)) mouse embryonic fibroblasts (MEFs). The regulatory role of Zmiz2 in Wnt-induced TCF/ß-catenin-mediated transcription can be restored in Zmiz2(-/-) MEFs that were infected with adenoviral expression vectors for Zmiz2. Moreover, enhancement of Zmiz2 on TCF/ß-catenin-mediated transcription was further demonstrated in Zmiz2 knockout and Axin2 reporter compound mice. Furthermore, the protein-protein interaction between ZMIZ2 and ß-catenin was identified by co-immunoprecipitation and in vitro protein pulldown assays. We also observed recruitment of endogenous ZMIZ2 onto the promoter region of the Axin 2 gene, a ß-catenin downstream target promoter, in a Wnt ligand-inducible manner. Finally, a promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz2 knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and ß-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways.

Mediator and SAGA have distinct roles in Pol II preinitiation complex assembly and function.

A key feature of RNA polymerase II (Pol II) preinitiation complexes (PICs) is their ability to coordinate transcription initiation with chromatin modification and remodeling. To understand how this coordination is achieved, we employed extensive proteomic and mechanistic analyses to study the composition and assembly of PICs in HeLa cell and mouse embryonic stem cell (ESC) nuclear extracts. Strikingly, most of the machinery that is necessary for transcription initiation on chromatin is part of the PIC. The PIC is nearly identical between ESCs and HeLa cells and contains two major coactivator complexes: Mediator and SAGA. Genome-wide analysis of Mediator reveals that it has a close correlation with Pol II, TATA-binding protein, and messenger RNA levels and thus may play a major role in PIC assembly. Moreover, Mediator coordinates assembly of the Pol II initiation factors and chromatin machinery into a PIC in vitro, whereas SAGA acts after PIC assembly to allow transcription on chromatin.

Polycomb repressive complex 1 (PRC1) disassembles RNA polymerase II preinitiation complexes.

Despite the important role of Polycomb in genome-wide silencing, little is known of the specific biochemical mechanism by which it inactivates transcription. Here we address how recombinant Polycomb repressive complex 1 (PRC1) inhibits activated RNA polymerase II preinitiation complex (PIC) assembly using immobilized H3K27-methylated chromatin templates in vitro. Recombinant PRC1 inhibited transcription, but had little effect on binding of the activator as reported previously. In contrast, Mediator and the general transcription factors were blocked during assembly or dissociated from preassembled PICs. Importantly, among the PIC components, Tata Binding Protein (TBP) was the most resistant to eviction by PRC1. Immobilized template experiments using purified PRC1, transcription factor II D (TFIID), and Mediator indicate that PRC1 blocks the recruitment of Mediator, but not TFIID. We conclude that PRC1 functions to block or dissociate PICs by interfering with Mediator, but leaves TBP and perhaps TFIID intact, highlighting a specific mechanism for PRC1 transcriptional silencing. Analysis of published genome-wide datasets from mouse embryonic stem cells revealed that the Ring1b subunit of PRC1 and TBP co-enrich at developmental genes. Further, genes enriched for Ring1b and TBP are expressed at significantly lower levels than those enriched for Mediator, TBP, and Ring1b. Collectively, the data are consistent with a model in which PRC1 and TFIID could co-occupy genes poised for activation during development.

Mediator coordinates PIC assembly with recruitment of CHD1.

Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remodeling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3- and CHD1-stimulated gene activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator. In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.

Requirement for deoxycytidine kinase in T and B lymphocyte development.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, "fine-tuning" role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.

LZTS2 is a novel beta-catenin-interacting protein and regulates the nuclear export of beta-catenin.

Beta-catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of beta-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3beta, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with beta-catenin, represses the transactivation of beta-catenin, and affects the subcellular localization of beta-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, beta-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear beta-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a beta-catenin-interacting protein that can modulate beta-catenin signaling and localization.

Transcriptional activation of the Staphylococcus aureus putP gene by low-proline-high osmotic conditions and during infection of murine and human tissues.

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 muM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.