Effects of small heat shock proteins on the thermal denaturation and aggregation of F-actin

Effect of recombinant chicken small heat shock protein with molecular mass 24 kDa (Hsp24) and recombinant human small heat shock protein with molecular mass 27 kDa (Hsp27) on the heat-induced denaturation and aggregation of skeletal F-actin was analyzed by means of differential scanning calorimetry and light scattering. All small heat shock proteins did not affect thermal unfolding of F-actin measured by differential scanning calorimetry, but effectively prevented aggregation of thermally denatured actin. Small heat shock protein formed stable complexes with denatured (but not with intact) F-actin. The size of these highly soluble complexes was smaller than the size of intact F-actin filaments. It is supposed that protective effect of small heat shock proteins on the cytoskeleton is at least partly due to prevention of aggregation of denatured actin.     

Some properties of complexes formed by small heat shock proteins with denaturated actin

The effects of a recombinant small heat shock protein with an apparent molecular weight of 27 kDa (Hsp27) and both wild type (Hsp27-wt) and S15D, S78D, S82D mutants (Hsp27-3D), which mimic the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin were studied. It has been shown that at a Hsp27/actin weight ration of 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal denaturation of F-actin, but efficiently prevent its further aggregation by forming soluble complexes with denatured actin. Formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. Hsp27-wt is known to exist as a high-molecular-weight oligomer, whereas Hsp27-3D forms only small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17–18 nm. On the other hand, the sedimentation coefficients of these complexes differed: they ranged 10–45 S in the case of Hsp27-3D and 18–60 S in case of Hsp27-wt. Thus, the initial oligomeric state of Hsp27 does not significantly affect its capacity to form small soluble complexes with denatured actin.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Purification and characterization of two small heat shock proteins from Anabaena sp. PCC 7120

Two small heat shock proteins (sHsps), Hsp17.8 and Hsp17.1, were identified in the cyanobacterium Anabaena sp. PCC 7120. Recombinant Hsp17.8 and Hsp17.1 were overexpressed in Escherichia coli and characterized here. Hsp17.8 was purified by sequential chromatography on DEAE-Sepharose and Superose 6 10/300 column, and Hsp17.1 was purified by Superose 6 10/300 column in 4M urea. Size exclusion chromatography demonstrated that both purified proteins form large oligomers approximately 420kDa and 410kDa, respectively. Both Hsp17.8 and Hsp17.1 showed chaperone-like activity to protect citrate synthase (CS) from thermal aggregation at 43 degrees C. Furthermore, both proteins were found to form complexes with denatured CS at 45 degrees C. Our study also demonstrated that despite a high degree of sequence homology and similar subunit size, Hsp17.1 showed higher hydrophobicity indicated by 8-anilino-1-naphthalene sulfonate fluorescence and thus greater chaperone-like activity. This is the first report of characterization and comparison of an sHsp system containing two chaperones in cyanobacteria.     

Some properties of human small heat shock protein Hsp20 (HspB6).

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa. At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial alpha-crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein. At pH 6.0, both alpha-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, alpha-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase. The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperature-dependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.     

Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure

A single monomeric porin, Omp-PA (37kDa), was isolated from the outer membrane of the gram-negative anaerobic rod Porphyromonas asaccharolytica. Further characterization revealed that this porin consists of two different fractions: a heat-modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with a molecular weight of 41kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. A liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the "closed" conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the "open" conformer of the protein. Cloning of the omp-PA gene revealed an open reading frame of 1161 bases, with a predicted protein sequence of 387 amino acids. The mature protein consists of 366 amino acids with a calculated MW of 41,102Da and an estimated pI of 7.24. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity with the OmpA consensus domain). Sequence comparison with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Digestion of the P. asaccharolytica outer membrane analysis of trypsin-digested Omp-PA yielded two proteins migrating with apparent molecular weights of 37 and 27kDa. These data fully supported our hypothesis that the C-terminal domain of the two-domain "closed" conformer of Omp-PA was digested by trypsin, whereas the single domain beta-barrel "open" conformer was inaccessible to trypsin.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

Some properties of complexes formed by small heat shock proteins with denatured actin

We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.     

Amino acid analogs while inducing heat shock proteins sensitize CHO cells to thermal damage

Amino acid analogs have been shown to induce heat shock proteins (HSPs). We have examined the effect of these analogs on the thermal sensitivity of Chinese hamster fibroblasts (HA-1) and their stable heat-resistant variants. We found that exposure of HA-1 cells and their heat-resistant variants to canavanine or L-azetidine-2-carboxylic acid cause enhanced synthesis of the three major mammalian HSPs (molecular weight 70,000, 87,000, and 110,000 kd). Although the synthesis of HSPs was increased, the analogs did not induce thermotolerance, a transient ability to protect cells from thermal damage. On the contrary, the analog treatment increased the thermal sensitivity of HA-1 cells, but not of the heat-resistant strains, when these cells were exposed subsequently to elevated temperatures. Our tentative explanation for these findings is that the incorporation of amino acid analogs into HSPs or other cellular proteins sensitizes HA-1 cells to heat. The heat-resistant strains contain higher levels of constitutive HSPs. The additional functional HSPs in the heat-resistant variants may protect these cells from thermal stress. The presence of some newly synthesized analog-substituted, perhaps nonfunctional, HSPs need not affect this thermal protection.     

The respective 27 kDa and 28 kDa protein kinase C substrates in vascular endothelial and MCF-7 cells are most probably heat shock proteins

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the phosphorylation of two distinct 27 kDa and 28 kDa proteins, respectively, in bovine vascular endothelial cells and in MCF-7 human breast cancer cells. These protein phosphorylation events were correlated to striking opposite cell growth responses to TPA, i.e., stimulation of vascular endothelial cell proliferation and inhibition of MCF-7 cell growth. Exposure of both vascular endothelial and MCF-7 cells to heat shock induced synthesis of the respective 27 kDa and 28 kDa proteins among a set of common and distinct other proteins as well as an increase in the degree of phosphorylation of the two 27 kDa and 28 kDa proteins. These results suggest that the two protein kinase C substrates very likely belong to the family of low molecular mass stress proteins.     

Protein products of the open reading frames encoding nonstructural proteins of human astrovirus serotype 8

Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.     

Small GTP-binding proteins in the nuclei of human placenta.

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [alpha(32)P]-GTP. Prior to this study the number of GTP-binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP-binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [alpha(32)P]-GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [alpha(32)P]-GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP-binding proteins. The GTP-binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP-binding proteins in the development and physiology of the placenta.     

Stress-induced aggregation profiles of GST-alpha-synuclein fusion proteins: role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability

Alpha-synuclein is a well-known heat-resistant protein that does not aggregate upon heat treatment, whereas glutathione S-transferase (GST) is a heat-labile protein that easily precipitates as a result of thermal stress. This paper reports the role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability. The region of alpha-synuclein that is responsible for the heat resistance was initially investigated using a series of deletion mutants, and the C-terminal acidic tail (residues 96-140) was found to be crucial for the thermosolubility of alpha-synuclein. The thermal behavior of the GST-alpha-synuclein fusion protein was next investigated, and the fusion protein was seen to be extremely heat-resistant. Using a series of GST-synuclein deletion mutants, the C-terminal acidic tail of alpha-synuclein was shown to play a critical role in conferring the heat resistance of the fusion proteins. Furthermore, the acidic tail appeared to protect the fusion protein from pH- and metal-induced protein aggregation, suggesting that the acidic tail can increase the virtual stability of the protein by protecting it from the aggregation induced by environmental stresses. Interestingly, the acidic tail also appeared to protect the GST enzyme from the thermal inactivation to a considerable extent. However, CD analysis of the heat-induced secondary structural changes of the GST-alpha-synuclein fusion protein revealed that the fusion protein is irreversibly denatured by heat treatment with a slightly lowered melting temperature (Tm). Thus, the results demonstrate that introducing an acidic tail into GST promotes the thermosolubility and virtual stability of the fusion protein, although it might be unfavorable for its intrinsic stability.     

Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

Evaluation of antigenic properties of Campylobacter jejuni and Campylobacter coli proteins in a western-immunoblot

Campylobacter jejuni and Campylobacter coli are the most common bacterial cause for acute diarrheal illnesses in developed countries. The aim of this study was to evaluate the antigenic properties of Campylobacterjejuni and Campylobacter coli proteins in western-blot assay. Whole-cell components of Campulobacter jejuni and Campylobacter coli were separated by sodium dodecyl sulfate-polyacrylamide gel electroforesis. Using this method we detected in all seven C. jejuni strains 21 peptides migrating between 180-29 kDa. All three Ccoli strains had a 17 bands migrating with the same molecular weight range. Proteins were transferred electrophoretically to nitrocellulose paper for immunoblotting experiments. The 74 kDa protein reacted strongly in all classes ofimmmunoglobulin with all tested human serum samples. We observed that this protein reacted also with human immunoglobulins for Salmonella and Yersinia sp. This cross-reaction observed for this protein could give false positive results in routine diagnosis of C. jejuni infections. The proteins with molecular weight of: 92, 62, 56, 52, 45-43, 29 kDa were most recognized in the 20 human serum samples. The other proteins of Cljejuni and C. coli, particularly in the 68-50 kDa and 45-31 kDa regions, were recognized occasionally and the response to these in reconvalescent sera was usually weak. The result of this study showed that the proteins with molecular weight: 92, 62, 56, 52, 45-43 and 29 kDa can be use in routine serological diagnostic of campylobacteriosis.     

Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components, 55 kDa and 43–40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.     

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.     

The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins.

We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.     

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I, indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca2(+)-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortin-like proteins with apparent Mr33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by tracheal gland cells cultured on microporous membrane of a double chamber culture system.     

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins.

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.