Biophysical characterization of ERp29. Evidence for a key structural role of cysteine 125

ERp29 is a major resident of the endoplasmic reticulum (ER) that seemingly plays an important role in most animal cells. Although a protein-folding association is widely supported, ERp29's specific molecular function remains unknown. A chaperone activity was postulated from evidence that ERp29 forms multimers like the classical ER chaperones, but conflicting results have emerged from our recent studies. Here a biophysical approach was used to clarify this issue and also reveal a key structural role for ERp29's characteristic cysteine, Cys-125. Applying hydrodynamic parameters derived from sedimentation and dynamic light-scattering analyses, a model of ERp29's quaternary structure was assembled from existing tertiary substructures. Comparison with Windbeutel, an ERp29-like protein from fruit fly with specialized chaperone activity, revealed similar tri-lobar gross structures but some finer differences consistent with functional divergence. Solubility and hydrophobic probe assays revealed moderate surface hydrophobicity, which was reduced in mutant ERp29 in which serine replaced Cys-125. This mutant was also relatively labile to proteolytic degradation, providing two reasons for the strict conservation of Cys-125. No multimerization was observed with untagged ERp29, which existed as tight homodimers (K(d) < 50 nm), whereas His-tagged ERp29 artifactually formed 670-kDa oligomers. These findings distinguish ERp29 biophysically from its peers in the ER including Windbeutel, endorsing our postulate that ERp29 adds a distinct type of folding activity to the ER machinery. By invoking novel functional associations for Cys-125 and the adjoining linker, new clues about how ERp29 might work have also arisen.     

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.     

The Alzheimer beta-peptide shows temperature-dependent transitions between left-handed 3-helix, beta-strand and random coil secondary structures

The temperature-induced structural transitions of the full length Alzheimer amyloid beta-peptide [A(beta)(1-40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 3(1) (polyproline II; PII)-helix at 0 degrees C to a random coil state at 60 degrees C, with an average DeltaH of 6.8 +/- 1.4 kJ.mol(-1) per residue, obtained by fitting a Zimm-Bragg model to the CD data. The transition is noncooperative for the shortest N-terminal fragment A(beta)(1-9) and weakly cooperative for A(beta)(1-40) and the longer fragments. By analysing the temperature-dependent 3J(HNH(alpha)) couplings and hydrodynamic radii obtained by NMR for A(beta)(1-9) and A(beta)(12-28), we found that the structure transition includes more than two states. The N-terminal hydrophilic A(beta)(1-9) populates PII-like conformations at 0 degrees C, then when the temperature increases, conformations with dihedral angles moving towards beta-strand at 20 degrees C, and approaches random coil at 60 degrees C. The residues in the central hydrophobic (18-28) segment show varying behaviour, but there is a significant contribution of beta-strand-like conformations at all temperatures below 20 degrees C. The C-terminal (29-40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of A(beta) may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.     

ERp29 regulates response to doxorubicin by a PERK-mediated mechanism

ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 −/− cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells' resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

Purification and structural characterization of the central hydrophobic domain of oleosin

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.     

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.     

Dimerization of ERp29, a PDI-like protein, is essential for its diverse functions

Protein disulfide isomerase (PDI)-like proteins act as oxido-reductases and chaperones in the endoplasmic reticulum (ER). How oligomerization of the PDI-like proteins control these activities is unknown. Here we show that dimerization of ERp29, a PDI-like protein, regulates its protein unfolding and escort activities. We have demonstrated previously that ERp29 induces the local unfolding of polyomavirus in the ER, a step required for viral infection. We now find that, in contrast to wild-type ERp29, a mutant ERp29 (D42A) that dimerizes inefficiently is unable to unfold polyomavirus or stimulate infection. A compensatory mutation that partially restores dimerization to the mutant ERp29 (G37D/D42A) rescues ERp29 activity. These results indicate that dimerization of ERp29 is crucial for its protein unfolding function. ERp29 was also suggested to act as an escort factor by binding to the secretory protein thyroglobulin (Tg) in the ER, thereby facilitating its secretion. We show that this escort function likewise depends on ERp29 dimerization. Thus our data demonstrate that dimerization of a PDI-like protein acts to regulate its diverse ER activities.     

Interrelation of conformational changes of crustacin and its capacity to bind specifically with carcino-embryonic antigen

Temperature-, ionic strength-, calcium ion- and pH-dependence of spatial structure of crustacin have been studied using CD and fluorescent spectroscopy. Secondary structure of crustacin was estimated by CD spectra. An irreversible conformational transition of crustacin's protein moiety connected with the loss of CEA-binding activity has been found at ca. 50 degrees C. Crustacin is shown to be calcium-binding protein, stability of the native crustacin conformation being markedly enhanced by calcium ions (1 mM Ca2+ shifted up the transition temperature by approximately 10 degrees C). Calcium binding and ionic strength increase led to alteration of both secondary and tertiary structures of crustacin. The highest CEA-binding activity was observed for the calcium-bond form of crustacin. A lack of specific interaction of crustacin with some saccharides was shown. Interrelation between conformation and immunochemical activity of crustacin is discussed.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Transport mechanisms in chloride channels.

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (less than 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50 degrees C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of alpha-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of alpha-helix content. Relatively large decreases in ellipticity were observed between 20 degrees C and 40 degrees C for FSR and 30 degrees C and 60 degrees C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.     

Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen

Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures.     

Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.     

Effect of cyclodextrin on the activity and secondary structure of horseradish peroxidase

The activity and secondary structure of horseradish peroxidase (HRP) was studied in aqueous solution containing alpha-, beta- and gamma-cyclodextrin (CD). The results showed that the activity of HRP was enhanced to different extents by the three kinds of CD. A Fourier Transform infrared (FTIR) spectroscopy study indicated that the amount of alpha-helical structure was important for the activity of HRP. This phenomenon is discussed.     

The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection

Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain.     

ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.     

A spectroscopic study of the temperature induced modifications on ferredoxin folding and iron-sulfur moieties

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of -helical content at relatively low temperatures (T(app)(m) approximately 44 degrees C), followed by the disruption of both iron-sulfur clusters (T(app)(m) approximately 53-60 degrees C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (T(app)(m) approximately 58 degrees C) and a change, rather than a significant net loss, of secondary structure (T(app)(m) approximately 60 degrees C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron-sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.     

Effect of proline to alanine mutation on the thermal stability of the all-beta-sheet protein tendamistat

The temperature dependent denaturation of wild-type tendamistat and of the proline-free triple mutant P7A/P9A/P50A was investigated using Fourier-transform infrared (FTIR) spectroscopy. Whereas the temperature-induced unfolding is reversible in the wild type, aggregation was observed for the proline-free tendamistat when studied under the same conditions. The midpoint unfolding temperature T(m) was found as 82.3+/-0.5 degrees C in (2)H2O. The thermal stability of the proline-free mutant is reduced by 15 degrees C as compared to the wild type. Changes in the strength of hydrogen bonding of tyrosine O-H groups upon unfolding and aggregation are reflected in small shifts of the C-C stretching mode of the aromatic ring near 1515 cm(-1). Evaluation of data from different infrared (IR) bands sensitive to changes in secondary structure as well as to changes in tertiary structure strongly supports a two-state model for the unfolding process of wild-type tendamistat.