Henry D. Herce

Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes

The recombinant HIV-1 Tat protein contains a small region corresponding to residues 47YGRKKRRQRR57R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions.

Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms.

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

Visualization and targeted disruption of protein interactions in living cells

Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species.

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1−/− cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

Chapter 5 Simulations of Temperature and Pressure Unfolding of Peptides and Proteins with Replica Exchange Molecular Dynamics

Publisher Summary Replica dynamics and its variations are effectively used to explore the folding kinetics of peptides and mini proteins. This chapter describes the use of replica exchange molecular dynamics (REMD) methods, the issues surrounding the use of REMD, its advantages, and limitations. The chapter also presents system-size limitations, the efficient selection of temperatures, the sampling of a canonical ensemble, thermodynamic equilibrium, the convergence of averages and discusses the extensions of the REMD that allows for the sampling density and and density states. Initial applications show that the REMD is able to quickly form secondary structural elements, such as alpha helices and beta hairpins. Further studies show that tens of nanoseconds are not sufficient to obtain the convergence of detailed free-energy surfaces and longer simulation times are required. It has been shown that for the Trp-cage mini-protein, the mean time for replicas to sample folding and unfolding basins is of the order of 20 ns, which implies that equilibrium calculations should be extended for many times this time. Unfortunately, these calculations, when done in explicit solvent models, require calculations that extend for months in todays computers.

The electrostatic surface term: (I) Periodic systems

The authors propose a new approach to understand the electrostatic surface contributions to the interactions of large but finite periodic distributions of charges. They present a simple method to derive and interpret the surface contribution to any electrostatic field produced by a periodic distribution of charges. They discuss the physical and mathematical interpretations of this term. They present several examples and physical details associated with the calculation of the surface term. Finally, they provide a simple derivation of the surface contribution to the virial. This term does not disappear even if tinfoil boundary conditions are applied.

Direct Homo- and Hetero-Interactions of MeCP2 and MBD2.

Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163–309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.