Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane

Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s)

The anaerobic (Class III) ribonucleotide reductase from Lactococcus lactis:Catalytic properties and allosteric regulation of the pure enzyme system

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase, Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli, The corresponding proteins, NrdD and NrdG, were purified close to homogeneity, The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E, coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated, Also the FeS cluster was easily lost from NrdG, The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effecters bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP, The two sites showed an unusually high degree of cooperativity with complex interactions between effecters and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.</p

The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-β peptide fibrils and inhibits secondary nucleation

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD β-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-β (Aβ42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aβ, the CTD constructs instead bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two β-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD

Big dynorphin is a neuroprotector scaffold against amyloid β-peptide aggregation and cell toxicity

Amyloid β-peptide (Aβ) misfolding into β-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aβ aggregation are highly relevant as possible AD treatments since they should protect against Aβ neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aβ40 the most abundant species of Aβ. Biophysical measurements indicate that Aβ40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aβ40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aβ40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.The authors would like to thank Mr. Jordi Pujols Pujol for skillful technical assistance in RP-HPLC experiments, and Mr. Mateo Calle Velásquez for skillful assistance in the docking process.Peer reviewe

In Silico Analysis of the Apolipoprotein E and the Amyloid β Peptide Interaction: Misfolding Induced by Frustration of the Salt Bridge Network

The relationship between Apolipoprotein E (ApoE) and the aggregation processes of the amyloid β (Aβ) peptide has been shown to be crucial for Alzheimer's disease (AD). The presence of the ApoE4 isoform is considered to be a contributing risk factor for AD. However, the detailed molecular properties of ApoE4 interacting with the Aβ peptide are unknown, although various mechanisms have been proposed to explain the physiological and pathological role of this relationship. Here, computer simulations have been used to investigate the process of Aβ interaction with the N-terminal domain of the human ApoE isoforms (ApoE2, ApoE3 and ApoE4). Molecular docking combined with molecular dynamics simulations have been undertaken to determine the Aβ peptide binding sites and the relative stability of binding to each of the ApoE isoforms. Our results show that from the several ApoE isoforms investigated, only ApoE4 presents a misfolded intermediate when bound to Aβ. Moreover, the initial α-helix used as the Aβ peptide model structure also becomes unstructured due to the interaction with ApoE4. These structural changes appear to be related to a rearrangement of the salt bridge network in ApoE4, for which we propose a model. It seems plausible that ApoE4 in its partially unfolded state is incapable of performing the clearance of Aβ, thereby promoting amyloid forming processes. Hence, the proposed model can be used to identify potential drug binding sites in the ApoE4-Aβ complex, where the interaction between the two molecules can be inhibited