Dhanashree D. Jagtap

Crystal structure of prostate secretory protein PSP94 shows an edge-to-edge association of two monomers to form a homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 A resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last beta-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.

Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

The prostate secretory protein of 94 amino acids (PSP94) has been shown to interact with cysteine-rich secretory protein 3 (CRISP-3) in human seminal plasma. Interestingly, PSP94 expression is reduced or lost in the majority of the prostate tumours, whereas CRISP-3 expression is upregulated in prostate cancer compared with normal prostate tissue. To obtain a better understanding of the individual roles these proteins have in prostate tumourigenesis and the functional relevance of their interaction, we ectopically expressed either PSP94 or CRISP-3 alone or PSP94 along with CRISP-3 in three prostate cell lines (PC3, WPE1-NB26 and LNCaP) and performed growth inhibition assays. Reverse transcription-polymerase chain reaction and Western blot analysis were used to screen prostate cell lines for PSP94 and CRISP-3 expression. Mammalian expression constructs for human PSP94 and CRISP-3 were also generated and the expression, localization and secretion of recombinant protein were assayed by transfection followed by Western blot analysis and immunofluorescence assay. The effect that ectopic expression of PSP94 or CRISP-3 had on cell growth was studied by clonogenic survival assay following transfection. To evaluate the effects of co-expression of the two proteins, stable clones of PC3 that expressed PSP94 were generated. They were subsequently transfected with a CRISP-3 expression construct and subjected to clonogenic survival assay. Our results showed that PSP94 and CRISP-3 could each induce growth inhibition in a cell line specific manner. Although the growth of CRISP-3-positive cell lines was inhibited by PSP94, growth inhibition mediated by CRISP-3 was not affected by the presence or absence of PSP94. This suggests that CRISP-3 may participate in PSP94-independent activities during prostate tumourigenesis.

Effect of FSH Receptor-Binding Inhibitor-8 on FSH-Mediated Granulosa Cell Signaling and Proliferation

Follicle‐stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle‐stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle‐stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non‐steroidal factors secreted by the ovaries are believed to modulate follicle‐stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle‐stimulating hormone receptor‐binding inhibitor‐8 purified from human follicular fluid has been extensively studied. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 has been shown to inhibit binding of follicle‐stimulating hormone to its receptor. The present article describes the effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on follicle‐stimulating hormone‐induced signaling in rat granulosa cells. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 inhibited the follicle‐stimulating hormone‐induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle‐stimulating hormone receptor‐binding inhibitor‐8 acts as a follicle‐stimulating hormone antagonist and affects the follicle‐stimulating hormone‐mediated signaling and proliferation in the granulosa cells.

Prostate Secretory Protein of 94 amino acids (PSP94) binds to prostatic acid phosphatase (PAP) in human seminal plasma.

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor, with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHRHB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHRHB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8D6A showed 50% lower IC50 compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8D6A interfered with the binding of FSH to its receptor.

Development of an ELISA for sPSP94 and utility of the sPSP94/sPSA ratio as a diagnostic indicator to differentiate between benign prostatic hyperplasia and prostate cancer.

BACKGROUND The serum PSA (sPSA) test has low specificity for prostate cancer (PCa), since sPSA also rises in benign prostatic hyperplasia (BPH). Serum PSP94 (sPSP94), a major secreted prostate protein, is indicated as a PCa marker. The potential of sPSP94 and sPSA in conjunction with each other to improve specificity of diagnostic test for PCa needs to be evaluated. METHODS PCa patients (n=33), BPH patients (n=44) and healthy controls (n=50) were recruited. A serum-based sandwich ELISA was developed to measure sPSP94 concentrations. Utility of sPSP94 in improving specificity of sPSA test was evaluated by studying sPSP94/sPSA ratios of study participants. RESULTS Considerable decrease in overlap among sPSP94/sPSA ratio values of BPH and PCa patients was observed, as compared to sPSP94 or sPSA alone. For differentiating between BPH and PCa patients, this ratio had a maximum area under the curve (AUC) of 0.859 (P=0.0132) and had a comparable sensitivity (90.91%) to sPSA with an increased specificity of 70.45%. Further, decision curve analysis (DCA) showed that sPSP94/sPSA ratio had a superior net benefit in identifying PCa, in patients opting for biopsy. CONCLUSION The sPSP94/sPSA ratio can be a better differentiating marker between BPH and PCa, than sPSP94 or sPSA alone.

Assessing safety and protein interactions of surface-modified iron oxide nanoparticles for potential use in biomedical areas

We have investigated the electrostatic interaction between bare iron oxide nanoparticles (IONPs) or low molecular weight chitosan coated iron oxide nanoparticles (LMWC-IONPs) and hen egg white lysozyme (HEWL) at different pH values using protein-nanoparticle reverse charge parity model. Physicochemical characterization of both IONPs and LMWC-IONPs were carried out using DLS, TEM, FE-SEM, XRD, TGA, XPS and VSM analysis. DLS, TEM and FE-SEM results indicated that both IONPs were monodispersed, with size ranging from 8 to 20nm. The coating of LMWC on IONPs was confirmed using zeta potential, TGA, XRD and XPS measurements. The cytotoxicity of both IONPs and LMWC-IONPs was studied in vitro in A549 human lung alveolar epithelial cells to assess their use in biomedical applications. Furthermore, the interactions between protein-nanoparticles were investigated by UV-visible, fluorescence and circular dichroism spectroscopic techniques. The present study suggests that water soluble LMWC surface modified IONPs are the promising nanomaterials. The safety and biocompatibility of these nanoparticles render them suitable for biomedical applications.

Molecular characterization of inhibin-A: Structure and expression analysis in Clarias batrachus

The inhibins are disulphide-linked heterodimeric glycoproteins that belong to the TGFβ superfamily. Inhibins have been well studied in mammals but the information about their structure and function is very limited in lower vertebrates. The aim of the present study was to characterize inhibin-A and to understand its receptor binding interaction, and to evaluate its biological function in Clarias batrachus. Structure prediction of inhibin-A revealed two glycosylation sites on inhibin-α (Asp262 and Asn334). Docking of inhibin-A with its receptor; betaglycan and Act RIIA showed that residues Ser321, Gly324 and Leu325 of inhibin-α are involved in high affinity binding with betaglycan while inhibin-βA bound to Act RIIA by forming hydrogen bonds. The mRNA transcript analysis of various tissues indicated the presence of higher to moderate expression of inhibin-α and inhibin-βA in the gonads and the extra-gonadal tissues. Further, stage specific expression showed decreased levels of inhibin-α in the gonads during the annual reproductive cycles. Inhibin-βA, activin-βB and Act RIIA increased in the brain during spawning while FSHr increased in the gonads during the preparatory phase. Our study provides molecular, structural and functional insights of inhibin-A for the first time in C. batrachus.

Crystallization and preliminary X-ray diffraction analysis of human seminal plasma protein PSP94.

The human seminal plasma protein PSP94 is a small protein of 94 residues that contains ten cysteines. Since its discovery about 25 years ago, several potential biological functions have been reported for this protein. Many PSP94 homologues have also been identified since then from various species, but no crystal structure has been determined to date. PSP94 has been purified from human seminal plasma and crystallized. These crystals diffracted to approximately 2.3 A resolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = 107.9, b = 107.9, c = 92.1 A. There are four molecules in the asymmetric unit. Structure solution by the heavy-atom method is currently in progress.

How the surface functionalized nanoparticles affect conformation and activity of proteins: Exploring through protein-nanoparticle interactions

To understand the effect of counter ions (Na+) on the secondary conformation and functionality of the lysozyme, we have studied the interaction of lysozyme with counterion associated iron oxide nanoparticles (IONPs). The investigation was carried out at pH 7.4 and 9.0, with three different types of NPs, namely, bare IONPs, low molecular weight chitosan modified IONPs (LMWC-IONPs) and the counterion (Na+) associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs) and confirmed by using various spectroscopy techniques. The difference in UV-vis absorbance (ΔA) between native and STP-LMWC-IONPs interacted hen egg white lysozyme (HEWL) was greater than that between native and NPs interacted HEWL at pH 9.0 compared with pH 7.4. Furthermore, STP-LMWC-IONPs exhibited quenching effect on lysozyme fluorescence spectrum at pH 9.0 due to binding of Na+ counterions to the protein, confirming denaturation of the latter. After HEWL interaction with STP-LMWC-IONPs (pH 9.0), CD spectra revealed a conformational change in the secondary structure of HEWL. Also, counterion induced lysozyme inactivation, due to interaction with nanoparticles at pH 9.0, was confirmed by enzymatic activity assay involving lysis of Micrococcus lysodeikticus. In conclusion, pH 9.0 was observed to be a more favorable condition, compared to pH 7.4, for the strongest electrostatic interaction between lysozyme and NPs. We postulate that the counterions in nanoparticle surface-coating can ameliorate protein misfolding or unfolding and also prevent their aggregation and, therefore, can be considered as a powerful and potential therapeutic strategy to treat incurable neurodegenerative disorders.