[Improving the biocompatibility of silicone implants using spider silk coatings: immunohistochemical analysis of capsule formation]. / Verbesserung der Biokompatibilität von Silikonimplantaten durch Spinnenseidenbeschichtung: Immunhistochemische Untersuchungen zum Einfluss auf die Kapselbildung.

INTRODUCTION: Optimisation of the biocompatibility of silicone implants and reduction of capsule formation around the surface of such implants are in the focus of plastic surgical biomaterial research. In addition to its extraordinary physical and biochemical properties, spider silk shows high biocompatibility. Therefore, the coating of silicone implant surfaces with recombinant spider silk was analysed regarding foreign body reactions. MATERIALS AND METHODS: In the context of a preclinical study, miniaturised silicone implants were implanted in the back of 60 Sprague-Dawley rats. The animals were randomised; 30 animals received a texturised implant coated with the recombinant spider silk protein eADF4(C16) and 30 animals received uncoated implants. 3, 6 and 12 months after implantation, implants together with the surrounding capsules were removed and submitted to histological and immunohistochemical assessment. RESULTS: Coating of silicone implants with the recombinant spider silk protein eADF4(C16) resulted in a delayed and significantly decreased foreign body reaction and a reduced capsule manifestation. CONCLUSION: eADF4(C16) seems to be a promising candidate for the reduction of foreign body-associated capsule formation. Moreover, coating of other medical implants with this recombinant spider silk protein may improve their biocompatibility with little additional effort.

Peptide adsorption on a hydrophobic surface results from an interplay of solvation, surface, and intrapeptide forces.

The hydrophobic effect, i.e., the poor solvation of nonpolar parts of molecules, plays a key role in protein folding and more generally for molecular self-assembly and aggregation in aqueous media. The perturbation of the water structure accounts for many aspects of protein hydrophobicity. However, to what extent the dispersion interaction between molecular entities themselves contributes has remained unclear. This is so because in peptide folding interactions and structural changes occur on all length scales and make disentangling various contributions impossible. We address this issue both experimentally and theoretically by looking at the force necessary to peel a mildly hydrophobic single peptide molecule from a flat hydrophobic diamond surface in the presence of water. This setup avoids problems caused by bubble adsorption, cavitation, and slow equilibration that complicate the much-studied geometry with two macroscopic surfaces. Using atomic-force spectroscopy, we determine the mean desorption force of a single spider-silk peptide chain as F = 58 +/- 8 pN, which corresponds to a desorption free energy of approximately 5 k(B)T per amino acid. Our all-atomistic molecular dynamics simulation including explicit water correspondingly yields the desorption force F = 54 +/- 15 pN. This observation demonstrates that standard nonpolarizable force fields used in classical simulations are capable of resolving the fine details of the hydrophobic attraction of peptides. The analysis of the involved energetics shows that water-structure effects and dispersive interactions give contributions of comparable magnitude that largely cancel out. It follows that the correct modeling of peptide hydrophobicity must take the intimate coupling of solvation and dispersive effects into account.

Protein aggregation as a cause for disease.

The ability of proteins to fold into a defined and functional conformation is one of the most fundamental processes in biology. Certain conditions, however, initiate misfolding or unfolding of proteins. This leads to the loss of functional protein or it can result in a wide range of diseases. One group of diseases, which includes Alzheimer's, Parkinson's, Huntington's disease, and the transmissible spongiform encephalopathies (prion diseases), involves deposition of aggregated proteins. Normally, such protein aggregates are not found in properly functioning biological systems, because a variety of mechanisms inhibit their formation. Understanding the nature of these protective mechanisms together with the understanding of factors reducing or deactivating the natural protection machinery will be crucial for developing strategies to prevent and treat these disastrous diseases.

The role of conformational flexibility in prion propagation and maintenance for Sup35p.

The [PSI(+)] factor of Saccharomyces cerevisiae is a protein-based genetic element (prion) comprised of a heritable altered conformation of the cytosolic translation termination factor Sup35p. In vitro, the prion-determining region (NM) of Sup35p undergoes conformational conversion from a highly flexible soluble state to structured amyloid fibers, with a rate that is greatly accelerated by preformed NM fiber nuclei. Nucleated conformational conversion is the molecular basis of the genetic inheritance of [PSI(+)] and provides a new model for studying amyloidogenesis. Here we investigate the importance of structure and structural flexibility in soluble NM. Elevated temperatures, chemical chaperones and certain mutations in NM increase or change its structural content and inhibit or enhance nucleated conformational conversion. We propose that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.

Bidirectional amyloid fiber growth for a yeast prion determinant.

The polymerization of many amyloids is a two-stage process initiated by the formation of a seeding nucleus or protofibril. Soluble protein then assembles with these nuclei to form amyloid fibers. Whether fiber growth is bidirectional or unidirectional has been determined for two amyloids. In these cases, bidirectional growth was established by time lapse atomic-force microscopy. Here, we investigated the growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p. The conformational changes in NM that lead to amyloid formation in vitro serve as a model for the self-perpetuating conformational changes in Sup35p that allow this protein to serve as an epigenetic element of inheritance in vivo. To assess the directionality of fiber growth, we genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled after fiber formation. The mutant protein assembled in vitro with kinetics indistinguishable from those of the wild-type protein and propagated the heritable genetic trait [PSI(+)] with the same fidelity. In reactions nucleated with prelabeled fibers, unlabeled protein assembled at both ends. Thus, NM fiber growth is bidirectional.

Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae.

The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.

The charged region of Hsp90 modulates the function of the N-terminal domain.

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.

The Hsp90 complex--a super-chaperone machine as a novel drug target.

Cells respond to sudden changes in the environmental temperature with increased synthesis of a distinct number of heat shock proteins (Hsps). Analysis of the function of these proteins in recent years has shown that all the major classes of conserved Hsps are molecular chaperones involved in assisting cellular protein folding and preventing irreversible side-reactions, such as unspecific aggregation. In addition to their function under stress conditions, molecular chaperones also play a critical role under physiological conditions. Hsp90 is one of the most abundant chaperones in the cytosol of eukaryotic cells. It is part of the cell's powerful network of chaperones to fight the deleterious consequences of protein unfolding caused by nonphysiological conditions. In the absence of stress, however, Hsp90 is an obligate component of fundamental cellular processes such as hormone signaling and cell cycle control. In this context, several key regulatory proteins, such as steroid receptors, cell cycle kinases, and p53, have been identified as substrates of Hsp90. Recently, Hsp90 was shown to be the unique target for geldanamycin, a potent new anti-tumor drug that blocks cell proliferation. Interestingly, under physiological conditions, Hsp90 seems to perform its chaperone function in a complex with a set of partner proteins, suggesting that the Hsp90 complex is a multi-chaperone machine specialized in guiding the maturation of conformationally labile proteins. The regulation of key signaling molecules of the cell by the Hsp90 machinery is a stimulating new concept emerging from these studies, and Hsp90 has become a promising new drug target.

Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence.

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.

S. cerevisiae and sulfur: a unique way to deal with the environment.

Saccharomyces cerevisiae is by far the best-studied unicellular eukaryote. Although yeast cells are very similar to higher eukaryotes in many respects, there is striking evidence that S. cerevisiae is not a perfect model for a eukaryotic cell (cf. 1). Here we report that yeast proteins contain a significantly lower amount of cysteine residues compared to other eukaryotes. Explanations for this phenomenon could not be found in the sulfur metabolism of yeast, which showed no major differences from other organisms (2-4). However, previous examinations could link a defect in sulfate uptake of S. cerevisiae to an increased resistance against toxic substances like selenate and chromate in the environment, which share the same permeases (5-7). This environmental problem might have caused S. cerevisiae to down-regulate its sulfate uptake and therefore lead to a lower amount of available sulfur in the cell, making it necessary to replace all dispensable sulfur amino acids in proteins. We show in two examples that S. cerevisiae proteins contain only such cysteine residues that are structurally or functionally needed. Therefore, we conclude that S. cerevisiae has solved a widespread environmental problem in a specific way which might be unique among eukaryotes.

ATP-binding properties of human Hsp90.

Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion. Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with half-maximal binding at 400 microM ATP was detected. The functional significance of this weak interaction remains enigmatic.

Assessment of the ATP binding properties of Hsp90.

Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90.