Volume 389, Issue 1

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.

The nightly bioluminescence of the dinoflagellate Gonyaulax is a circadian rhythm caused by the presence in cells of specialized bioluminescent organelles, termed scintillons, containing the reaction catalyst luciferase, the substrate luciferin and a luciferin-binding protein (LBP). LBP levels increase at the start of the night phase because of increased protein synthesis rates in vivo , and this regulation has been ascribed to circadian binding of an inhibitory protein factor binding to the 3′ untranslated region (UTR) of lbp mRNA at times when LBP is not normally synthesized. To purify and characterize the binding factor, the electrophoretic mobility shift assays and UV crosslinking experiments used to first characterize the factor were repeated. However, neither these protocols nor binding to biotinylated RNA probes confirmed the presence of a specific circadian RNA-binding protein. Furthermore, neither RNA probe screening of a cDNA library expressed in bacteria nor three-hybrid assays in yeast were successful in isolating a cDNA encoding a protein able to bind specifically to the lbp 3′UTR. Taken together, these results suggest that alternative mechanisms for regulating lbp translation should now be examined.

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 μ m . Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.

The specificity of bacterial nutrient importers of the ATP-binding cassette (ABC) type depends on external receptor proteins that not only bind the solute to be transported, but also initiate the transport process by inducing ATP hydrolysis in the cytoplasmic nucleotide-binding domains. Here we propose a mode of ligand binding to the solute-binding protein AatJ that is required for glutamate uptake by the AatJMQP transporter in Pseudomonas putida KT2440. A homology model of the AatJ-glutamate complex was constructed using the E. coli glutamine-binding protein GlnH as the template. The general validity of the model was then confirmed by alanine scanning mutagenesis of several residues predicted to interact with the ligand and by semi-quantitative binding studies with [ 14 C]-Glu and [ 14 C]-Asp. A database search indicated that AatJ is a member of a distinct subfamily of the family 3 solute-binding proteins with specificity towards glutamate and aspartate.

Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130 Cas phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130 Cas protein levels, we cotransfected cells with 1 n m p130 Cas siRNA to partially reduce p130 Cas protein to control levels. Although p130 Cas Tyr(P) 249 phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130 Cas increased cell spreading by 20% compared to p130 Cas siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130 Cas . FAK-binding mutant SH3 domain-deleted rat p130 Cas was not phosphorylated after adhesion and, unlike full-length p130 Cas , did not restore spreading after human-specific p130 Cas siRNA knockdown of endogenous p130 Cas . Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130 Cas phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130 Cas .

The p53 tumour suppressor gene is frequently mutated in human tumours and different tumour-derived mutations have varying effects on cells. The effect of a novel tumour-derived p53 mutation and two recently described mutations from South African breast cancer patients on the growth rate, colony formation, cell cycle arrest after irradiation and response to chemotherapeutic drugs was investigated. None of the p53 mutations had any significant effect on the inherent growth rate of the cells; however, contact inhibition of growth in two of the mutants was lost. These same two mutants formed colonies in soft agar, whereas the third mutant did not. All three of the mutants failed to show a G 1 cell cycle arrest after exposure to 7 Gy of [ 60 Co] radiation, albeit to different degrees. Cells expressing the p53 mutants were either more sensitive to cisplatin and melphalan or more resistant than the untransfected cells, depending on the mutation. However, there was no difference in response to daunorubicin treatment. These results demonstrate that different p53 mutations exert varying biological effects on normal cells, with some altering checkpoint activation more effectively than others. The data also suggest that the nature of the p53 mutation influences the sensitivity to cytostatic drugs.

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for the production of human recombinant GPCRs. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoter pufQ . We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host – R. sphaeroides – to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.

A healthy vascular endothelium is coated by the endothelial glycocalyx. Its main constituents are transmembrane syndecans and bound heparan sulphates. This structure maintains the physiological endothelial permeability barrier and prevents leukocyte and platelet adhesion, thereby mitigating inflammation and tissue oedema. Heparinase, a bacterial analogue to heparanase, is known to attack the glycocalyx. However, the exact extent and specificity of degradation is unresolved. We show by electron microscopy, immunohistological staining and quantitative measurements of the constituent parts, that heparinase selectively sheds heparan sulphate from the glycocalyx, but not the syndecans.

The 27-mer peptide CP1B-[1–27] derived from exon 1B of calpastatin stands out among the known inhibitors for μ- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4–23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1–27]. Starting with this peptide, the turn-like region Glu 10 (i)-Leu 11 (i+1)-Gly 12 (i+2)-Lys 13 (i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4–23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu 10 -Lys 13 upon binding to μ-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

A cDNA encoding insulin-degrading enzyme (IDE) was cloned from tomato ( Solanum lycopersicum ) and expressed in Escherichia coli in N-terminal fusion with glutathione S -transferase. GST- Sl IDE was characterized as a neutral thiol-dependent metallopeptidase with insulinase activity: the recombinant enzyme cleaved the oxidized insulin B chain at eight peptide bonds, six of which are also targets of human IDE. Despite a certain preference for proline in the vicinity of the cleavage site, synthetic peptides were cleaved at apparently stochastic positions indicating that Sl IDE, similar to IDEs from other organisms, does not recognize any particular amino acid motif in the primary structure of its substrates. Under steady-state conditions, an apparent K m of 62±7 μ m and a catalytic efficiency ( k cat / K m ) of 62±15 m m -1 s -1 were determined for Abz-SKRDPPKMQTDLY(NO 3 )-NH 2 as the substrate. GST- Sl IDE was effectively inhibited by ATP at physiological concentrations, suggesting regulation of its activity in response to the energy status of the cell. While mammalian and plant IDEs share many of their biochemical properties, this similarity does not extend to their function in vivo , because insulin and the β-amyloid peptide, well-established substrates of mammalian IDEs, as well as insulin-related signaling appear to be absent from plant systems.