Extracellular vesicle docking at the cellular port: Extracellular vesicle binding and uptake.

Extracellular vesicles (EVs), lipid bilayer-enclosed structures that contain a variety of biological molecules shed by cells, are increasingly becoming appreciated as a major form of cell-to-cell communication. Indeed, EVs have been shown to play important roles in several physiological processes, as well as diseases such as cancer. EVs dock on to the surfaces of recipient cells where they transmit signals from the cell surface and/or transfer their contents into cells to elicit functional responses. EV docking and uptake by cells represent critical, but poorly understood processes. Here, we focus on the mechanisms by which EVs dock and transfer their contents to cells. Moreover, we highlight how these findings may provide new avenues for therapeutic intervention.

Structural characterization of semen coagulum-derived SEM1(86-107) amyloid fibrils that enhance HIV-1 infection.

SEM1(86-107) is a 22-residue peptide corresponding to residues 86-107 in the semenogelin I protein. SEM1(86-107) is an abundant component of freshly liquefied semen and forms amyloid fibrils capable of enhancing HIV infection. To probe the factors affecting fibril formation and gain a better understanding of how differences in pH between semen and vaginal fluid affect fibril stability, this study determined the effect of pH on SEM1(86-107) fibril formation and dissociation. The SEM1(86-107) fibril structure (i.e., residues that comprise the fibrillar core) was also probed using hydrogen-deuterium exchange mass spectrometry (HDXMS) and hydroxyl radical-mediated protein modification. The average percent exposure to hydroxyl radical-mediated modification in the SEM1(86-107) fibrils was determined without requiring tandem mass spectrometry spectral acquisition or complete separation of modified peptides. It was found that the residue exposures calculated from HDXMS and hydroxyl radical-mediated modification were similar. These techniques demonstrated that three regions of SEM1(86-107) comprise the amyloid fibril core and that positively charged residues are exposed, suggesting that electrostatic interactions between SEM1(86-107) and HIV or the cell surface may be responsible for mediating HIV infection enhancement by the SEM1(86-107) fibrils.

Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy.

PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression and purification of uniformly (13)C- and (15)N-labeled PAPf39 peptide, through expression as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH.

Core sequence of PAPf39 amyloid fibrils and mechanism of pH-dependent fibril formation: the role of monomer conformation.

PAPf39, a 39-residue peptide fragment from human prostatic acidic phosphatase, has been shown to form amyloid fibrils in semen (SEVI), which increase HIV infectivity by up to 5 orders of magnitude. The sequence of the PAPf39 fibrillar core was identified using hydrogen-deuterium exchange (HDX) mass spectrometry and protease protection assays. The central and C-terminal regions are highly protected from HDX and proteolytic cleavage and, thus, are part of the fibrillar core. Conversely, the N-terminal region is unprotected from HDX and proteolytic cleavage, suggesting that it is exposed and not part of the fibrillar core. This finding was tested using two N-terminal truncated variants, PAPf39Δ1-8 and PAPf39Δ1-13. Both variants formed amyloid fibrils at neutral pH. However, these variants showed a markedly different pH dependence of fibril formation versus that of PAPf39. PAPf39 fibrils can form at pH 7.7, but not at pH 5.5 or 2.5, while both N-terminally truncated variants can form fibrils at these pH values. Thus, the N-terminal region is not necessary for fibril formation but modulates the pH dependence of PAPf39 fibril formation. PAPf39Δ1-8 and PAPf39Δ1-13 are capable of seeding PAPf39 fibril formation at neutral pH, suggesting that these variants are structurally compatible with PAPf39, yet no mixed fibril formation occurs between the truncated variants and PAPf39 at low pH. This suggests that pH affects the PAPf39 monomer conformational ensemble, which is supported by far-UV circular dichroism spectroscopy. A conceptual model describing the pH dependence of PAPf39 aggregation is proposed and provides potential biological implications.

Mechanism of fibril formation by a 39-residue peptide (PAPf39) from human prostatic acidic phosphatase.

PAPf39 is a 39-residue peptide fragment from the sequence of human prostatic acidic phosphatase. This peptide was shown to form amyloid-like fibrils, which have been implicated in facilitating semen-mediated HIV transmission. Thus understanding molecular details of PAPf39 peptide fibril formation may aid in elucidating the mechanism of how PAPf39 fibrils are involved in HIV etiology. To this end, the kinetics of PAPf39 peptide fibrillization was studied using a battery of biophysical methods (atomic force microscopy, ThT fluorescence assays, far-UV circular dichroism spectroscopy, deep-UV resonance Raman spectroscopy, size exclusion chromatography, analytical ultracentrifugation, and small-angle X-ray scattering). It has been shown that fibril formation follows a nucleation-dependent elongation mechanism. Several critical factors for fibrillization have been identified. It was shown that agitation and/or seeding is required for fibril formation at 37 degrees C and neutral pH, with an additional requirement of a salt concentration above approximately 100 mM. Fibril formation by the PAPf39 peptide is inhibited by low pH or by low salt concentration at neutral pH. These observations suggest that the nucleation and fibrillization of the PAPf39 peptide are a tug-of-war between the interactions formed upon agitation and the electrostatic interactions, modulated by pH and salt concentration.

Intramolecular diffusion controls aggregation of the PAPf39 peptide.

The 39-residue fragment of human prostatic acidic phosphatase (PAP) is found in high concentrations in semen and easily form fibrils. Previous work has shown that fibrillization is accelerated with a deletion of the first 8, mostly charged residues and it was hypothesized that fibrillization depended on the dynamics of these peptides. To test this hypothesis we have measured the intramolecular diffusion of the full length and 8-residue deletion peptides at two different pHs and found a correlation with fibrillization lag time. These results can be explained by a simple kinetic model of the early stages of aggregation in which oligomerization is controlled by the rate of peptide reconfiguration.