Mitochondrial protein synthesis in vitro is not an artifact

It is definitively shown that the D variant of bovine β-lactoglobulin D differs from the B variant in having a substitution Gln → Glu which involves residue 45 in the complete sequence recently published by Braunitzer et al. [4].     

Core protein I as an artifact in complex III.

SDS electrophoresis gels of complex III from yeast mitochondria were run after incubation of the enzyme at several different temperatures. It was found that the intensity of the more slowly moving core protein band was substantially affected by the incubation temperature. Four low molecular weight polypeptides were eluted from gels electrophoresed after preincubation of the enzyme at 15° C for 12 hours. These polypeptides were then incubated for 5 minutes at 100° followed by SDS gel electrophoresis. A polypeptide with the same molecular weight as the anomalous core protein was resolved.     

Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

BackgroundVitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5′-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the “inactive” vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of “harsh” extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms.MethodsWe designed a “mild” extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays.ResultsA “mild” extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual “harsh” protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl.ConclusionsOur procedure reveals a high content of MeCbl in rat liver.General significanceThis result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.     

The generation of neurons and oligodendrocytes from a common precursor cell

We have used a recombinant retrovirus carrying the lacZ gene to study the developmental potential of precursor cells from the embryonic rat cerebral cortex in dissociated cell culture. Virus was used to label a small number of cultured cells genetically so that their fate could be determined. Infected clones were detected with an anti-beta-galactosidase serum, and the labeled cells were identified using monoclonal antibodies. The results revealed that most precursor cells generated a single cell type, the majority being either neurons or oligodendrocytes. However, a proportion of the neuronal clones also included oligodendrocytes. This proportion increased until embryonic day 16 when 18% of the neuronal clones were of this type. This suggests that during neurogenesis in the cerebral cortex there exists a cell with the potential to generate these two quite different neural cell types.     

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.     

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.     

The mitochondrial permeability transition from in vitro artifact to disease target

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to approximately 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.     

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation

Amyloid precursor protein (APP) is the precursor of amyloid β (Aβ) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aβ peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aβ peptides Aβ_1–40, Aβ_1–42 and Aβ_25–35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aβ peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aβ peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aβ_1–42 and Aβ_25–35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aβ under static condition but not platelet aggregation, and is responsible for Aβ-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aβ peptides accumulate in cerebral vessel walls or atherosclerotic plaques.     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin‐binding disulfide‐constrained loop region of human ß‐amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C‐terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL‐37, these peptides efficiently killed the Gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram‐positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop‐structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

The carboxy-terminal peptides of in vitro precursor and in vivo mature kappa light chains are identical.

Protein structural analysis of several precursor light chains indicates that these in vitro products include a cysteine-containing tryptic peptide that is indistinguishable from the carboxy-terminal tryptic tripeptide present in in vivo mature kappa light chains. It is concluded that although in vitro precursor light chains contain an additional amino-terminal sequence, their carboxy-terminus is the same as that found in in vivo mature light chains.     

The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell‐surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over‐expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition.

     

In vitro prothrombin synthesis from a purified precursor protein I. Development of a bovine liver cell-free system

Purified PIVKA-II is converted into prothrombin by a cell-free system derived from normal bovine liver.The reaction conditions are described.