A change of mitochondrial membrane permeability is essential for apoptosis, leading to translocation of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. We recently showed that the Bcl-2 family of proteins regulate cytochrome c release and the mitochondrial membrane potential (Δψ) by directly modulating the activity of the voltage-dependent anion channel (VDAC) through binding. Here we investigated the biochemical role of the conserved N-terminal homology domain (BH4) of Bcl-x L , which has been shown to be essential for inhibition of apoptosis, with respect to the regulation of mitochondrial membrane permeability and found that BH4 was required for Bcl-x L to prevent cytochrome c release and Δψ loss. A study using VDAC liposomes revealed that Bcl-x L , but not Bcl-x L lacking the BH4 domain, inhibited VDAC activity. Furthermore, BH4 oligopeptides of Bcl-2 and Bcl-x L , but not mutant peptides, were able to inhibit both VDAC activity on liposomes even in the presence of Bax and apoptotic Δψ loss in isolated mitochondria. It was also shown that the BH4 domain, fused to the protein transduction domain of HIV TAT protein (TAT-BH4), efficiently prevented apoptotic cell death. These results indicate that the BH4 of Bcl-2/Bcl-x L is essential and sufficient for inhibiting VDAC activity, which in turn prevents apoptotic mitochondrial changes, and for preventing apoptotic cell death. Finally, the data suggest that the TAT-BH4 peptide is potentially useful as a therapeutic agent for diseases caused by accelerated apoptosis.

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock -factor 32 encoded by the rpoH gene. Increased 32 levels result from both enhanced synthesis and stabilization. Previous work indicated that 32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of 32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNA f Met complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPAR␥. Here, we report that some PPAR␥ ligands, including 15-deoxy-⌬ 12,14 -prostaglandin J 2 , covalently bind to a cysteine residue in the PPAR␥ ligand binding pocket through a Michael addition reaction by an ␣,␤-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPAR␥, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the ␣,␤-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the ␣,␤-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPAR␥. We propose that PPAR␥ senses oxidation of fatty acids by recognizing such an ␣,␤-unsaturated ketone as a common moiety.

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

Translocated in liposarcoma (TLS) is an importantprotein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113 Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 ؋ 10 ؊5 M, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.The translocated in liposarcoma (TLS) 1 protein, also termed FUS, was first identified in human myxoid and round cell liposarcomas as an oncogenic fusion protein with a stressinduced DNA-binding transcription factor, CCAAT enhancerbinding homologous protein (CHOP, also known as GADD153 or DDIT3) (1, 2). The resultant fusion protein (TLS-CHOP), consisting of the N-terminal half of TLS and the full-length CHOP, appears to act as a potent transcription factor possibly by combining the TLS transactivation activity and the CHOP DNA binding activity. A different type of fusion protein, TLS-ERG (a member of the erythroblast transformation-specific (ETS) family of transcription factors), was subsequently detected in human acute myeloid leukemia (3).The normal TLS, consisting of 526 amino acids with a calculated molecular mass of 53 kDa, belongs to a family including the closely related proteins Ewing's sarcoma (EWS) (4) and TAF II 68 (TATA-binding protein-associated factor) (5). Thus, they are collectively called the TET (TLS, EWS, TAF II 68) family. EWS and TAF II 68 interact with components of the RNA polymerase II complex (5, 6). Moreover sarcoma-associated RNA-binding fly homologue (SARFH), a Drosophila homologue of TLS, is colocalized with the polymerase on active chromatin (7). The N-terminal domain of TLS is involved in transcription...