Mechanistic and structural studies on amyloid fibrils found in semen that enhance HIV infectivity

Abstract

In particular, the kinetics of PAPf39 fibril formation 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 was shown that PAPf39 fibril formation follows a nucleation-dependent elongation mechanism and several critical factors necessary for fibril formation were identified. Agitation and/or seeding are necessary for fibril formation at neutral pH, with an additional requirement of a salt concentration above ~ 100 mM. PAPf39 fibril formation is inhibited by low pH or by low salt concentration at neutral pH.

Importantly, these studies provide insight into the factors driving amyloid fibril formation, the mechanism of PAPf39 and SEM1(86-107) amyloid fibril formation, and fibril-mediated enhancement of viral infectivity. These studies demonstrate common properties between two semen-derived amyloid fibrils, specifically the pH dependence of amyloid fibril formation and dissociation and similarities in exposed residues in the fibril structures.

In addition to PAPf39, SEM1(86-107) fibrils were extensively characterized to determine commonalities between semen-derived amyloid fibrils and elucidate common mechanisms of fibril-mediated viral infectivity enhancement. Therefore, the pH dependence of SEM1(86-107) fibril formation and dissociation were probed via thioflavin T assays and atomic force microscopy. Similar to PAPf39, SEM1(86-107) fibril formation and dissociation are dependent on pH: fibrils form at pH 7.7, but not at pH 5.5 or lower and preformed fibrils fully dissociate at pH 2.5, but not at pH 5.5. Furthermore, the SEM1(86-107) fibril core sequence was determined using hydrogen-deuterium exchange mass spectrometry and hydroxyl radical-mediated peptide modification. As in PAPf39 fibrils, it appears that positively charged residues are exposed on the fibril surface and may be responsible for enhancing HIV infectivity.

The colloidal properties of the PAPf39 fibrils and its variants were also studied. Although the truncated PAPf39 variants and full length PAPf39 have similar morphology according to AFM imaging and seeding assays, they have markedly different colloidal properties. Therefore, the surface charge (zeta potential) and sedimentation of the PAPf39 fibrils and its variants were characterized. In particular, it was found that the zeta potential (surface charge) correlates with the sedimentation of the PAPf39 fibrils and its variants.

Furthermore, 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. 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. The truncated variants 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.

Protein aggregation is a common phenomenon that occurs in a wide variety of biological systems. Protein aggregation can occur at almost any step in the protein folding process, during long term storage, at high protein concentrations (e.g. inclusion bodies), and under a variety of conditions, including a wide range of temperatures, pH values, and ionic strengths. There are two common types of protein aggregates: amorphous aggregates and amyloid fibrils. Amorphous aggregates appear to lack a well-defined structure, while amyloid fibrils have well-defined fibrillar morphology and beta-sheet structure. Both types of aggregates appear to represent generic, stable protein conformations that can be adopted by any protein, implying that there is an underlying mechanism driving aggregation. Fundamentally, protein aggregation studies aim to answer the following question: what forces drive protein aggregation? Overall, protein aggregation is of great interest, not only because of its fundamental importance, but because it is a major issue hindering the use of proteins as biomedical therapeutics and natural catalysts in industry. Moreover, amyloid fibrils are involved in many protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's disease, type II diabetes, prion diseases, and various amyloidoses.

More specifically, these studies probed these questions via extensive biophysical characterization of two peptides involved in semen-derived amyloid fibril-mediated enhancement of HIV infectivity: PAPf39, a 39 residue peptide fragment of human prostatic acidic phosphatase corresponding to residues 248-286, and SEM1(86-107), a 22 residue peptide corresponding to residues 86-107 of the human semenogelin I protein.

Although many amyloid fibrils are involved in neurodegenerative diseases, numerous peptides form amyloid fibrils in semen which increase HIV and other retrovirus infectivity. Interestingly, this effect seems to be limited to the fibrillar state of the peptides since little to no infectivity enhancement is observed when cells are exposed to the non-fibrillar form of the peptides. Since sexual transmission accounts for approximately 80% of new HIV infections and semen markedly enhances HIV infection rates in vitro, semen-derived amyloid fibrils that increase HIV infectivity may be targets for therapeutic development. This study aims to determine the mechanism of fibril formation; the structure of the amyloid fibrils; the interactions that occur between semen-derived amyloid fibrils, HIV, and the cell surface; and the mechanism of fibril-mediated enhancement of viral infectivity. The ultimate goal of these studies is to develop therapeutics capable of ameliorating fibril-mediated enhancement of HIV infectivity.