In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.

Small ubiquitin-like modifier (SUMO) 1 is a protein of 97 amino acids that is structurally similar to ubiquitin and has been called by other names including Smt3p, Pmt2p, PIC-1, GMP1, Ubl1, and Sentrin (1). Like ubiquitin, SUMO has been found to be covalently attached to certain lysine residues of specific target proteins (2). In contrast to ubiquitination, however, sumoylation does not promote the degradation of proteins but instead alters a number of different functional parameters of proteins, depending on the protein substrate in question. These parameters include but are not limited to properties such as subcellular localization, protein partnering, and DNA-binding and/or transactivation functions of transcription factors (2-4). The contrast between the functional effects of ubiquitination and sumoylation is most striking in the case of IB, where sumoylation stabilizes the protein by modifying the same residue that is ubiquitinated, thereby directly competing with that pathway (5). This review will focus on the regulation of SUMO modification and its role in controlling the functional properties of proteins. The reader is also referred to other excellent reviews on this topic (2-4, 6 -8). Enzymology and Regulation of SUMO Conjugationand Deconjugation Three different ubiquitous SUMO-related proteins have been identified in mammalian cells, SUMO-1, SUMO-2, and SUMO-3, with SUMO-2 and SUMO-3 having greater sequence relatedness with each other than with 4). Recently a tissue-specific SUMO-4 has been identified in human kidney with homology to SUMO-2/3, which raises the possibility that some SUMO proteins could have tissue-dependent functions (9). SUMO modification occurs on the lysine in the consensus sequence ⌿KXE (where ⌿ represents a hydrophobic amino acid, and X represents any amino acid) (2, 3). The mechanism involved in maturation and transfer of SUMO to target substrates is very similar to that seen with ubiquitination and other ubiquitin-like proteins (3, 4). This process involves four enzymatic steps: maturation, activation, conjugation, and ligation ( Fig. 1). In the first step the SUMO protein is cleaved by SUMO-specific carboxyl-terminal hydrolase to produce a carboxyl-terminal diglycine motif. This process of maturation is identical with all three mammalian SUMO forms. After maturation, SUMO proteins are able to be utilized for conjugation to proteins. The SUMO-activating (E1) enzyme is a heterodimer consisting of Aos1 and Uba2 (also known as SAE1/SAE2 or Sua1/hUba2 in humans). Activation of SUMO by the E1 is an ATP-dependent process and results in the formation of a thioester bond between SUMO and the Uba2 subunit of the E1-activating enzyme. Activation is followed by transfer of SUMO from the E1 enzyme to a conserved cysteine in the conjugating (E2) enzyme, Ubc9. This single E2 enzyme identified so far for the sumoylation pathway contrasts with the multiple E2 enzymes involved in attaching ubiquitin to proteins (4, 10).The final step of sumoylation involves ligation of SUMO to the target protei...

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 μM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 μM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42°C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39°C vs 42°C), within the physiological range of fever. In this study, we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h, whereas HSF1−/− T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1−/− mice at fever temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle progression under the stressful conditions encountered during fever.

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear poreassociating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear poreassociating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.The up-regulation of heat shock proteins such as HSP70 that occurs in response to exposure to elevated temperature and many other stress conditions is vital for the ability of cells to survive these stresses. Because of their crucial cytoprotective function, it is very important that up-regulation of HSP expression after stress occur as rapidly and as efficiently as possible. An intriguing finding of past studies is that stress conditions inhibit the export of many mRNAs from the nucleus, but mRNAs encoding heat shock proteins continue to be efficiently exported during stress (1-7). However, the mechanism by which HSP mRNAs bypass this stress-associated export inhibition was not known.HSF1 is the transcription factor responsible for up-regulating transcription of HSP70 and other genes in response to elevated temperature and other stress conditions. HSF1 performs this function by undergoing stress-induced trimerization to the DNA-binding form and then interacting with heat shock elements in the promoters of these genes to increase their transcription (8,9). TPR is a 270-kDa polypeptide that is associated with the nuclear basket on the nucleoplasmic face of the nuclear pore complex (10 -17). Previous data suggest that TPR is involved in the export of both mRNAs and proteins from the nucleus (13, 16, 18 -20). During the course of yeast two-hybrid experiments in our laboratory, we identified the existence of an interaction between HSF1 and the TPR protein. The results presented here suggest that the HSF1-TPR interaction could be important for the export of HSP mRNAs during stress conditions.