Fragments Of Prostatic Acid Phosphatase Research Articles

The Extracellular Molecular Chaperone Clusterin Inhibits Amyloid Fibril Formation and Suppresses Cytotoxicity Associated with Semen-Derived Enhancer of Virus Infection (SEVI).

Clusterin is a glycoprotein present at high concentrations in many extracellular fluids, including semen. Its increased expression accompanies disorders associated with extracellular amyloid fibril accumulation such as Alzheimer’s disease. Clusterin is an extracellular molecular chaperone which prevents the misfolding and amorphous and amyloid fibrillar aggregation of a wide variety of unfolding proteins. In semen, amyloid fibrils formed from a 39-amino acid fragment of prostatic acid phosphatase, termed Semen-derived Enhancer of Virus Infection (SEVI), potentiate HIV infectivity. In this study, clusterin potently inhibited the in vitro formation of SEVI fibrils, along with dissociating them. Furthermore, clusterin reduced the toxicity of SEVI to pheochromocytoma-12 cells. In semen, clusterin may play an important role in preventing SEVI amyloid fibril formation, in dissociating SEVI fibrils and in mitigating their enhancement of HIV infection.

Rapid Formation of Peptide/Lipid Coaggregates by the Amyloidogenic Seminal Peptide PAP248-286

Protein/lipid coassembly is an understudied phenomenon that is important to the function of antimicrobial peptides as well as the pathological effects of amyloid. Here, we study the coassembly process of PAP248-286, a seminal peptide that displays both amyloid-forming and antimicrobial activity. PAP248-286 is a peptide fragment of prostatic acid phosphatase and has been reported to form amyloid fibrils, known as semen-derived enhancer of viral infection (SEVI), that enhance the viral infectivity of human immunodeficiency virus. We find that in addition to forming amyloid, PAP248-286 much more readily assembles with lipid vesicles into peptide/lipid coaggregates that resemble amyloid fibrils in some important ways but are a distinct species. The formation of these PAP248-286/lipid coaggregates, which we term “messicles,” is controlled by the peptide:lipid (P:L) ratio and by the lipid composition. The optimal P:L ratio is around 1:10, and at least 70% anionic lipid is required for coaggregate formation. Once formed, messicles are not disrupted by subsequent changes in P:L ratio. We propose that messicles form through a polyvalent assembly mechanism, in which a critical surface density of PAP248-286 on liposomes enables peptide-mediated particle bridging into larger species. Even at ∼50-fold lower PAP248-286 concentrations, messicles form at least 10-fold faster than amyloid fibrils. It is therefore possible that some or all of the biological activities assigned to SEVI, the amyloid form of PAP248-286, could instead be attributed to a PAP248-286/lipid coaggregate. More broadly speaking, this work could provide a potential framework for the discovery and characterization of nonamyloid peptide/lipid coaggregates by other amyloid-forming proteins and antimicrobial peptides.

Membrane Disruption and Peptide/Lipid Co-Assembly by the Amyloid-Forming Peptide, PAP248-286

Both amyloid-forming and antimicrobial peptides (AMPs) are membrane active, displaying a variety of membrane-disrupting and -remodeling activities including membrane leakage, vesicle fusion, and vesicle aggregation. The cytotoxicity of membrane disrupting pre-fibrillar oligomers underlies the pathological activity of amyloid-forming peptides. Conversely, the disruption and aggregation of microbial membranes by AMPs is critical to their function in an innate immune response. Therefore, studying the factors driving the membrane activity of these peptides may be valuable in understanding both normal function and diseased states. We focus our studies on an amyloidogenic fragment of prostatic acid phosphatase (PAP248-286). In its amyloid state, this peptide is termed “semen-derived enhancer of viral infection” (SEVI), and greatly increases HIV infectivity. It is also thought to play functional roles in the antimicrobial activity of seminal fluid and sperm clearance during fertilization. Using liposomes as model membranes and biophysical tools including negative stain electron microscopy, dynamic light scattering, and single particle fluorescence burst analysis, we have demonstrated that PAP248-286 displays dramatic membrane remodeling activities, as it can form large, fibrillar lipid-peptide co-aggregates that are distinct from SEVI amyloid and form much more readily. We have also found that PAP248-286 is a potent membrane disruptor. Using fluorescence dequenching experiments we have determined that PAP248-286 disrupts liposomes in a charge and dose dependent manner. We have also developed new approaches to improve the fitting and analysis of complex time-correlated single photon counting (TCSPC) experimental data, which has allowed us to characterize its leakage mechanism. Our work to elucidate the determinants of PAP248-286 membrane activity is not only important for understanding its biological activities, but also provides a framework for studying the membrane activity of other biologically important amyloid-forming and antimicrobial peptides.

Investigating Effects and Determinants of PAP248–286‐Membrane Interactions: Membrane Leakage and Peptide/Lipid Co‐Aggregation

Many amyloid‐forming peptides appear to possess potentially beneficial antimicrobial activities. Furthermore, it has been suggested that this previously unrecognized activity may have a role in innate immunity. Antimicrobial peptides, which are important effectors in the innate immune response, can act either directly by inducing membrane leakage and cytolysis, or indirectly through bacterial adhesion inhibition or enhanced phagocytosis via bacterial agglutination. Amyloid‐forming peptides themselves display a variety of membrane‐disrupting and membrane‐remodeling activities, including membrane leakage and vesicle aggregation. In turn, some membranes are known to catalyze amyloid formation. Peptide‐membrane interactions may thus underlie both the pathological activity of amyloid‐forming peptides through their cytotoxicity due to pre‐fibrillar oligomers and their beneficial functions through their direct or indirect antimicrobial activities. Therefore, studying the factors driving the interactions between amyloid‐forming peptides and membranes may be valuable in understanding their roles in both healthy and diseased states.To understand the general principles that govern the interactions of amyloid‐forming peptides with membranes, we use liposomes as model membranes to gain precise control over their size and composition. We have also chosen to focus our studies on an amyloidogenic fragment of prostatic acid phosphatase (PAP248–286). In its amyloid state, this peptide is named “semen‐derived enhancer of viral infection” (SEVI), and greatly increases HIV infectivity. Using spectroscopic techniques, we have discovered that PAP248–286 causes liposome disruption in a charge and dose dependent manner. This dependence is expected given the cationic nature of PAP248–286, and is consistent with similar activities of other pre‐amyloid toxins and antimicrobial peptides. In addition, we have discovered that PAP248–286 forms large, fibrillar lipid‐peptide co‐aggregates. These co‐aggregates have only been observed at specific peptide to lipid ratios with completely anionic liposomes. While this species, which we have termed “messicles”, looks somewhat fibrillar and is Thioflavin T positive, the morphology and kinetics of formation are more reminiscent of an amorphous aggregate than bona fide amyloid. We propose that messicle formation occurs via a mechanism wherein addition of the right amount of negatively charged liposomes completely or partially neutralizes the positive charges on PAP248–286, allowing coated liposomes to self‐associate, and eventually leading to flocculate formation. Since this process would be controlled by the binding affinity of the peptides for the liposomes, messicles might be able to form by this mechanism with other membrane compositions. Considering messicles as another structure in the diverse repertoire of states accessible to PAP248–286 may be important to understanding its function. As amyloid formation and amorphous aggregation occur for other disease‐associated peptides as well, this work could also guide further investigation into their membrane activity and potentially beneficial functions.Support or Funding InformationNSF Graduate Research Fellowship to E.V.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High throughput virtual screening and in silico ADMET analysis for rapid and efficient identification of potential PAP248-286 aggregation inhibitors as anti-HIV agents

Human semen is principal vehicle for transmission of HIV-1 and other enveloped viruses. Several endogenous peptides present in semen, including a 39-amino acid fragments of prostatic acid phosphatase (PAP248-286) assemble into amyloid fibrils named as semen-derived enhancer of viral infection (SEVI) that promote virion attachment to target cells which dramatically enhance HIV virus infection by up to 105-fold. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound, is the major catechin found in green tea which disaggregates existing SEVI fibers, and inhibits the formation of SEVI fibers. The aim of this study was to screen a number of relevant polyphenols to develop a rational approach for designing PAP248-286 aggregation inhibitors as potential anti-HIV agents. The molecular docking based virtual screening results showed that polyphenolic compounds 2–6 possessed good docking score and interacted well with the active site residues of PAP248-286. Amino acid residues of binding site namely; Lys255, Ser256, Leu258 and Asn265 are involved in binding of these compounds. In silico ADMET prediction studies on these hits were also found to be promising. Polyphenolic compounds 2–6 identified as hits may act as novel leads for inhibiting aggregation of PAP248-286 into SEVI.

Epigallocatechin-3-gallate rapidly remodels PAP85-120, SEM1(45-107), and SEM2(49-107) seminal amyloid fibrils.

ABSTRACTSemen harbors amyloid fibrils formed by proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) that potently enhance HIV infectivity. Amyloid but not soluble forms of these peptides enhance HIV infection. Thus, agents that remodel these amyloid fibrils could prevent HIV transmission. Here, we confirm that the green tea polyphenol, epigallocatechin-3-gallate (EGCG), slowly remodels fibrils formed by PAP248-286 termed SEVI (semen derived enhancer of viral infection) and also exerts a direct anti-viral effect. We elucidate for the first time that EGCG remodels PAP85-120, SEM1(45-107), and SEM2(49-107) fibrils more rapidly than SEVI fibrils. We establish EGCG as the first small molecule that can remodel all four classes of seminal amyloid. The combined anti-amyloid and anti-viral properties of EGCG could have utility in preventing HIV transmission.

Repurposing Hsp104 to Antagonize Seminal Amyloid and Counter HIV Infection

Naturally occurring proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) form amyloid fibrils in seminal fluid, which capture HIV virions and promote infection. For example, PAP248-286 fibrils, termed SEVI (semen-derived enhancer of viral infection), can potentiate HIV infection by several orders of magnitude. Here, we design three disruptive technologies to rapidly antagonize seminal amyloid by repurposing Hsp104, an amyloid-remodeling nanomachine from yeast. First, Hsp104 and an enhanced engineered variant, Hsp104(A503V), directly remodel SEVI and PAP85-120 fibrils into non-amyloid forms. Second, we elucidate catalytically inactive Hsp104 scaffolds that do not remodel amyloid structure, but cluster SEVI, PAP85-120, and SEM1(45-107) fibrils into larger assemblies. Third, we modify Hsp104 to interact with the chambered protease ClpP, which enables coupled remodeling and degradation to irreversibly clear SEVI and PAP85-120 fibrils. Each strategy diminished the ability of seminal amyloid to promote HIV infection, and could have therapeutic utility.

Spatial Structures of PAP(262–270) and PAP(274–284), Two Selected Fragments of PAP(248–286), an Enhancer of HIV Infectivity

Prostatic acid phosphatase (PAP) assembles into amyloid fibrils that facilitate infection by HIV. Its peptide fragments PAP(248–286) and PAP(85–120) also enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of monomeric PAP(248–286) in a biomembrane-mimicking environment can be separated into an N-terminal unordered region, an α-helical central domain, and an α/310-helical C-terminal section (Nanga et al., J. Am. Chem. Soc., 131:17972–17979, 2009). In this work, we used two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy techniques to study spatial structures of isolated central [PAP(262–270)] and C-terminal [PAP(274–284)] fragments of PAP(248–286) in SDS micelle solutions. NMR studies revealed the formation of complexes of both peptides with SDS micelles, with attraction to the micelle membranes occurring mainly through nonpolar and uncharged residues of the peptides. We demonstrate that, when interacting with SDS micelles, PAP(262–270) and PAP(274–284) form α-helical and 310-helical secondary structures, respectively, similar to that found previously for the 39-residue PAP(248–286).

Semen-derived amyloid fibrils affect spermatozoa motility and fertilization

Semen contains amyloid fibrils derived from fragments of prostatic acid phosphatase (PAP) and semenogelins (SEMs) that potently increase human immunodeficiency virus infection1,2. The purpose of this study was to evaluate the effect of seminal amyloids on spermatozoa function.

Interaction of prostatic acid phosphatase fragments with a lipid bilayer as studied by NMR spectroscopy

The effects of five fragments of prostatic acid phosphatase on dimyristoylphosphatidylcholine lipid multi-lamellar liposomes were studied by 2H and 31P NMR spectroscopy and those on planar supported multi-bilayers of the same lipid, by 1H and 31P NMR spectro-scopy. It was found that hydrophobic interaction is a dominated factor of the peptide–membrane binding, while the short α-helical fragments PAP(262-270) and PAP(262-272) strongly interact with the membrane at the interface, generally following to the Gibbs free energy of water-to-interface insertion.

Semen-Derived Enhancer of Viral Infection (SEVI) Binds Bacteria, Enhances Bacterial Phagocytosis by Macrophages, and Can Protect against Vaginal Infection by a Sexually Transmitted Bacterial Pathogen

The semen-derived enhancer of viral infection (SEVI) is a positively charged amyloid fibril that is derived from a self-assembling proteolytic cleavage fragment of prostatic acid phosphatase (PAP(248-286)). SEVI efficiently facilitates HIV-1 infection in vitro, but its normal physiologic function remains unknown. In light of the fact that other amyloidogenic peptides have been shown to possess direct antibacterial activity, we investigated whether SEVI could inhibit bacterial growth. Neither SEVI fibrils nor the unassembled PAP(248-286) peptide had significant direct antibacterial activity in vitro. However, SEVI fibrils bound to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Neisseria gonorrhoeae) bacteria, in a charge-dependent fashion. Furthermore, SEVI fibrils but not the monomeric PAP(248-286) peptide promoted bacterial aggregation and enhanced the phagocytosis of bacteria by primary human macrophages. SEVI also enhanced binding of bacteria to macrophages and the subsequent release of bacterially induced proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-1β). Finally, SEVI fibrils inhibited murine vaginal colonization with Neisseria gonorrhoeae. These findings demonstrate that SEVI has indirect antimicrobial activity and that this activity is dependent on both the cationic charge and the fibrillar nature of SEVI.

Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences.

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1−40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed.

147Characterizing the conformational space of two disordered peptides in different solutions

Amyloid fibrils formed by peptides found in semen have been shown to enhance HIV infectivity in vitro. The first of these peptides to be identified was the 248–286 fragment of prostatic acid phosphatase (PAP248–286) (Munich et al., 2007). PAP248–286 is highly cationic, and its fibrils might facilitate infection by decreasing the electrostatic repulsion between the negatively charged surfaces of the virus and the target cell. Whereas PAP248–286 can easily form fibrils in seminal fluid, it needs rapid agitation in other environments, and certain ions have been shown to be critical for its assembly into fibrils (Olsen et al., 2012). However, mutation of the positively charged residues to alanine results in a peptide (PAP248–286Ala) that can more easily form fibrilar aggregates. We studied PAP248–286 and PAP248–286Ala fibril formation in water and water + NaCl environments. While PAP248-286Ala can efficiently form fibrils in both water and water + NaCl, PAP248-286 can only do so in a water + NaCl solution. The inability of PAP248–286 to form fibrils in water could be due solely to repulsion between the positively charged peptides, an effect that might be diminished by the presence of salt. However, it is also possible that the explanation lies in PAP248–286’s failure to populate conformations that can easily lead to ordered aggregates. To answer this question, using molecular dynamics simulations, we characterized the ensemble of conformations populated by the two peptides in water and water + NaCl environments. The results indicate that PAP248-286Ala favors contacts that stabilize a strand-turn-strand, or β-arch, motif around P31, the only proline residue in the sequence. Because β-arches are a common feature in amyloid fibrils, and because it is very unlikely that a proline residue would be in any position other than the β-arch, we expect the formation of this motif to be the rate-limiting step in PAP248–286Ala / PAP248–286 fibril formation. Moreover, the contacts stabilizing the β-arch would bring positively charged residues into contact in PAP248–286, which, consistent with the experimental results, would be facilitated by the presence of negative ions. To summarize, we have tried to understand if the inability of PAP248–286 to efficiently form fibrils in water is only due to a slower aggregation caused by electrostatic repulsion between the positively charged peptides. Our data suggest that this effect is also due to electrostatic repulsion between the residues within each monomeric peptide, which prevents PAP248–286 from populating conformations that would lead to ordered aggregates.

Inhibition of semen-derived enhancer of virus infection (SEVI) fibrillogenesis by zinc and copper

Semen-derived enhancer of virus infection (SEVI), a naturally occurring peptide fragment of prostatic acid phosphatase, enhances HIV infectivity by forming cationic amyloid fibrils that aid the fusion of negatively charged virion and target cell membranes. Cu(II) and Zn(II) inhibit fibrillization of SEVI in a kinetic assay using the fibril-specific dye ThT. TEM suggests that the metals do not affect fibril morphology. NMR shows that the metals bind to histidines 3 and 23 in the SEVI sequence. ITC experiments indicate that SEVI forms oligomeric complexes with the metals. Dissociation constants are micromolar for Cu(II) and millimolar for Zn(II). Because the Cu(II) and Zn(II) concentrations that inhibit fibrillization are comparable with those found in seminal fluid the metals may modulate SEVI fibrillization under physiological conditions.

The Surprising Role of Amyloid Fibrils in HIV Infection.

Despite its discovery over 30 years ago, human immunodeficiency virus (HIV) continues to threaten public health worldwide. Semen is the principal vehicle for the transmission of this retrovirus and several endogenous peptides in semen, including fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2), assemble into amyloid fibrils that promote HIV infection. For example, PAP248-286 fibrils, termed SEVI (Semen derived Enhancer of Viral Infection), potentiate HIV infection by up to 105-fold. Fibrils enhance infectivity by facilitating virion attachment and fusion to target cells, whereas soluble peptides have no effect. Importantly, the stimulatory effect is greatest at low viral titers, which mimics mucosal transmission of HIV, where relatively few virions traverse the mucosal barrier. Devising a method to rapidly reverse fibril formation (rather than simply inhibit it) would provide an innovative and urgently needed preventative strategy for reducing HIV infection via the sexual route. Targeting a host-encoded protein conformer represents a departure from traditional microbicidal approaches that target the viral machinery, and could synergize with direct antiviral approaches. Here, we review the identification of these amyloidogenic peptides, their mechanism of action, and various strategies for inhibiting their HIV-enhancing effects.

Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI

BackgroundHIV-1 is usually transmitted in the presence of semen. We have shown that semen boosts HIV-1 infection and contains fragments of prostatic acid phosphatase (PAP) forming amyloid aggregates termed SEVI (semen-derived enhancer of viral infection) that promote virion attachment to target cells. Despite its importance for the global spread of HIV-1, however, the effect of semen on virus infection is controversial.ResultsHere, we established methods allowing the meaningful analysis of semen by minimizing its cytotoxic effects and partly recapitulating the conditions encountered during sexual HIV-1 transmission. We show that semen rapidly and effectively enhances the infectivity of HIV-1, HIV-2, and SIV. This enhancement occurs independently of the viral genotype and coreceptor tropism as well as the virus producer and target cell type. Semen-mediated enhancement of HIV-1 infection was also observed under acidic pH conditions and in the presence of vaginal fluid. We further show that the potency of semen in boosting HIV-1 infection is donor dependent and correlates with the levels of SEVI.ConclusionsOur results show that semen strongly enhances the infectivity of HIV-1 and other primate lentiviruses and that SEVI contributes to this effect. Thus, SEVI may play an important role in the sexual transmission of HIV-1 and addition of SEVI inhibitors to microbicides may improve their efficacy.

Identification of gp17 glycoprotein and characterization of prostatic acid phosphatase (PAP) and carboxypeptidase E (CPE) fragments in a human seminal plasma fraction interacting with concanavalin A.

The decapacitating fraction of human seminal plasma, which strongly interacts with concanavalin A, is constituted by high mannose-type N-linked glycoproteins, most of them of less than 44 kDa. Each component with apparent molecular mass of 30, 18, and 17 kDa respectively, as judged by SDS-PAGE, was submitted to "in gel" digestion with trypsin followed by HPLC separation of the peptides and sequencing. They were characterized at microscale as gp17, an aspartyl protease that possibly contributes to liquefaction of the seminal plasma coagulum, two fragments of human acid phosphatase (17 and 30 kDa, respectively), and a 17-kDa fragment of carboxypeptidase E. Neither the fragments of prostatic acid phosphatase nor that of carboxypeptidase E had been described before in the human seminal fluid. Very weak bands, of apparent molecular masses 44 and 52 kDa, are consistent with presence of small amounts of parent compounds, prostatic acid phosphatase and carboxypeptidase E.

Heparin-binding proteins of human seminal plasma homologous with boar spermadhesins

Protein homologues to boar seminal plasma spermadhesins with the N-terminal sequence AQN (AQN spermadhesins) and with the N-terminal sequence AWN (AWN spermadhesins) were detected in human seminal plasma and characterized. They were isolated as heparin-binding (HB) proteins from human seminal plasma by affinity chromatography on heparin–Sepharose and then separated into 12 fractions (HB1–HB12) by RP HPLC or into four major fractions (HB-I–HB-IV) by gel filtration. Rabbit antibody against boar seminal plasma AQN 1 spermadhesin cross-reacted with 10–14 kDa proteins of fraction HB7, and antibody against AWN 1 spermadhesin cross-reacted with 11–14 kDa proteins of fractions HB9 and HB11. Both antibodies interacted with 10–14 kDa proteins in fractions HB-I and HB-II. The N-terminal amino acid sequence 1AQNKG 5… was determined in the 14 kDa protein of fraction HB-I cross-reacting with AQN 1 antibodies. A component detected among 10–14 kDa proteins of HB7 cross-reacting with rabbit antiserum against AQN 1 had the N-terminal sequence 1GELKFVTLVFAVGDYE 16, which is similar to the sequence of a fragment of prostatic acid phosphatase. Lactoferrin and its fragments were immunodetected with rabbit antibody against human milk lactoferrin in fractions HB7–HB11. This was proved by N-terminal sequencing of a lactoferrin fragment immunodetected in fraction HB7. N-terminal amino acid sequence analysis of the dominant component of fraction HB2 revealed the presence of a fragment of semenogelin I.