Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Structural details of an interaction between cardiolipin and an integral membrane protein

Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid–protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data collected over the resolution range 30.0–2.1 Å show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase, promotes dissociation of αs from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of αs from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant αs prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated αs (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound αs, but not αs· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of αs and that dissociation of αs·GTP from βγ is likely to mediate receptor-induced αs depalmitoylation and translocation of the protein to cytosol in intact cells.

The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization

Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR–AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR–AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.

Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression.

Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized

Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

Globular cluster ages

We review two new methods to determine the age of globular clusters (GCs). These two methods are more accurate than the classical isochrone fitting technique. The first method is based on the morphology of the horizontal branch and is independent of the distance modulus of the globular cluster. The second method uses a careful binning of the stellar luminosity function and determines simultaneously the distance and age of the GC. We find that the oldest galactic GCs have an age of 13.5 ± 2 gigayears (Gyr). The absolute minimum age for the oldest GCs is 10.5 Gyr (with 99% confidence) and the maximum 16.0 Gyr (with 99% confidence). Therefore, an Einstein–De Sitter Universe (Ω = 1) is not totally ruled out if the Hubble constant is about 65 ± 10 Km s−1 Mpc−1.

Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.