Hsp70 Molecular Chaperone Facilitates Endoplasmic Reticulum-associated Protein Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Yeast

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29–231). Using Cu2+/ascorbate, we oxidized SHaPrP(29–231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29–101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 μM. Concomitant with oxidation, SHaPrP(29–231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37°C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X

The γ-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca2+-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.

Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation

We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, “active” dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247–267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.

Characterization of Spermidine Binding to Solubilized Plasma Membrane Proteins from Zucchini Hypocotyls1

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 μm, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1 : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases1

Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.

C/EBPɛ mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut)

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPɛ) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPɛ activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPɛ−/− mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBPɛ and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBPɛ in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBPɛ mRNA. We therefore hypothesize that C/EBPɛ positively regulates SGP gene expression, and that C/EBPɛ is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBPɛ promoter is regulated by CDP/cut during myeloid differentiation.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP–CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.

We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.