Coexpression of wild-type and mutant prion proteins alters their cellular localization and partitioning into detergent-resistant membranes

Transmissible spongiform encephalopathies (TSEs) are a group of diseases of infectious, sporadic and genetic origin, found in higher organisms and caused by the pathological form of the prion protein. The inheritable subgroup of TSEs is linked to insertional or point mutations in the prion gene prnp , which favour its misfolding and are passed on to offspring in an autosomal-dominant fashion. The large majority of patients with these diseases are heterozygous for the prnp gene, leading to the coexpression of the wild-type (wt) (PrP^C) and the mutant forms (PrPmut) in the carriers of these mutations. To mimic this situation in vitro , we produced Fischer rat thyroid cells coexpressing PrPwt alongside mutant versions of mouse PrP including A117V, E200K and T182A relevant to the human TSE diseases Gestmann–Sträussler–Scheinker (GSS) disease and familial Creutzfeldt–Jakob disease (fCJD). We found that coexpression of mutant PrP with wt proteins does not affect the glycosylation pattern or the biochemical characteristics of either protein. However, FRET and co-immunoprecipitation experiments suggest an interaction occurring between the wt and mutant proteins. Furthermore, by comparing the intracellular localization and detergent-resistant membrane (DRM) association in single- and double-expressing clones, we found changes in the intracellular/surface ratio and an increased sequestration of both proteins in DRMs, a site believed to be involved in the pathological conversion (or protection thereof) of the prion protein. We, therefore, propose that the mutant forms alter the subcellular localization and the membrane environment of the wt protein in co-transfected cells. These effects may play a role in the development of these diseases.     

Conformational transformation and selection of synthetic prion strains

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.     

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.     

Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

Conformational analysis of polypeptides and proteins for the study of protein folding, molecular recognition, and molecular design

Conformational energy calculations provide an understanding as to how interatomic interactions lead to the three-dimensional structures of polypeptides and proteins, and how these molecules interact with other molecules. Illustrative results of such calculations pertain to model systems (-helices and -sheets, and interactions between them), to various open-chain and cyclic peptides, to fibrous proteins, to globular proteins, and to enzyme-substrate complexes. In most cases, the validity of the computations is established by experimental tests of the predicted structures.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985. This paper first appeared in the Israel Journal of Chemistry, Vol. 27, 1986.     

Conformational energy analysis of the leucine repeat regions of C/EBP, GCN4, and the proteins of the myc, jun, and fos oncogenes

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and themyc, jun, andfos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long -helical regions of the polypeptide chains in a configuration termed the leucine zipper. In this paper, conformational energy analysis is used to determine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic -helix with the leucine side-chains arrayed on one side in such a way to favor leucine zipper dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed leucine zipper configuration as a critical structural feature of this class of DNA binding proteins.     

Common structural traits across pathogenic mutants of the human prion protein and their implications for familial prion diseases

Human (Hu) familial prion diseases are associated with about 40 point mutations of the gene coding for the prion protein (PrP). Most of the variants associated with these mutations are located in the globular domain of the protein. We performed 50 ns of molecular dynamics for each of these mutants to investigate their structure in aqueous solution. Overall, 1.6 μs of molecular dynamics data is presented. The calculations are based on the AMBER(parm99) force field, which has been shown to reproduce very accurately the structural features of the HuPrP wild type and a few variants for which experimental structural information is available. The variants present structural determinants different from those of wild-type HuPrP and the protective mutation HuPrP(E219K-129M). These include the loss of salt bridges in α₂-α₃ regions and the loss of π-stacking interactions in the β₂-α₂ loop. In addition, in the majority of the mutants, the α₃ helix is more flexible and Y169 is more solvent exposed. The presence of similar traits in this large spectrum of mutations hints to a role of these fingerprints in their known disease-causing properties. Overall, the regions most affected by disease-linked mutations in terms of structure and/or flexibility are those involved in the pathogenic conversion to the scrapie form of the protein and in the interaction with cellular partners. These regions thus emerge as optimal targets for antibody- and ligand-binding studies.     

Bone protects proteins over thousands of years: extraction, analysis, and interpretation of extracellular matrix proteins in archeological skeletal remains

In a good state of preservation, bone conserves the entire protein pattern of extracellular bone matrix proteins over thousands of years. The quality of the profiles of matrix proteins isolated from ancient bones (ranging from the pre-Pottery Neolithic Phase to Early Modern Times from different archaeological sites in different geographical areas), separated by electrophoresis, is as good as those from recent bones. Molecules arising from collagenous proteins (e.g., collagen type I), from the noncollagenous group (e.g., osteonectin), and from the immune system (e.g., immunoglobulin G) were identified in Western blots by specific antibodies. A comparative study of the immunoglobulin G content of the bones of five prehistoric children showed the lowest immunoglobulin G content in a child who suffered from chronic scurvy. Ancient bone proteins were also separated by two-dimensional electrophoresis. This technique makes fractionation of the complex protein mixtures of extracellular bone matrix more reproducible. Bone retains a chemical memory of earlier metabolic stimuli in its configuration of collagenous and noncollagenous proteins. In combination with the results of the microscopic examination of ancient bone, it should be possible to obtain more reliable information on the history and the evolution of diseases, based on analysis of intact proteins.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.