MOESM1 of Antiproliferative and apoptotic effects of black turtle bean extracts on human breast cancer cell line through extrinsic and intrinsic pathway

Additional file 1: Figure S1. PI staining assay showing the dead cells when treated with BTB extract (50 and 100 ¾g/ml) in MCF-7 and MDA-MB231 cells, respectively

Studies on the interactions of SAP-1 (an N-terminal truncated form of cystatin S) with its binding partners by CD-spectroscopic and molecular docking methods

<div><p>SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of β-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.</p></div

Human Epididymis Protein-4 (HE-4): A Novel Cross-Class Protease Inhibitor

<div><p>Epididymal proteins represent the factors necessary for maturation of sperm and play a crucial role in sperm maturation. HE-4, an epididymal protein, is a member of whey acidic protein four-disulfide core (WFDC) family with no known function. A WFDC protein has a conserved WFDC domain of 50 amino acids with eight conserved cystine residue. HE-4 is a 124 amino acid long polypeptide with two WFDC domains. Here, we show that HE-4 is secreted in the human seminal fluid as a disulfide-bonded homo-trimer and is a cross-class protease inhibitor inhibits some of the serine, aspartyl and cysteine proteases tested using hemoglobin as a substrate. Using SPR we have also observed that HE-4 shows a significant binding with all these proteases. Disulfide linkages are essential for this activity. Moreover, HE-4 is N-glycosylated and highly stable on a wide range of pH and temperature. Taken together this suggests that HE-4 is a cross-class protease inhibitor which might confer protection against microbial virulence factors of proteolytic nature.</p> </div

Searching new targets to counter drug resistance – GTPase-Obg mRNA expression analysis in <i>Mycobacterium</i> under stress and <i>in silico</i> docking with GTPase inhibitors

Searching new targets to counter drug resistance – GTPase-Obg mRNA expression analysis in <i>Mycobacterium</i> under stress and <i>in silico</i> docking with GTPase inhibitor

SPR sensograms of HE-4 interaction with papain and pepsin.

<p>Concentrations of HE-4 were fixed at 10 µM. Concentrations of papain and pepsin were 75 nM. Injection time of papain and pepsin is indicated by an arrow. (<b>A</b>) Pepsin-HE-4 interaction. (<b>B</b>) Papain-HE-4 interaction.</p

SPR sensograms of HE-4 interaction with serine proteases.

<p>HE-4 immobilised on CM5 chip as described in methods. Concentration of HE-4 was fixed at 10 µM. Concentrations of serine proteases (from top to bottom) were 300, 150, and 75 nM respectively. (<b>A</b>) Trypsin-HE-4 interaction. (<b>B</b>) Chymotrypsin-HE-4 interaction. (<b>C</b>) PSA- HE-4 interaction. (<b>D</b>) Proteinase K- HE-4 interaction.</p

Purification of HE-4 protein from human seminal plasma using affinity, ion exchange and gel filtration chromatography.

<p>(<b>A</b>) Elution profile of anion exchanger DEAE sephacel. The peaks formed as a function of X-axis as elution volume in ml and Y-axis is mA at 280 nm. The second (….) line represents the gradient of NaCl. The first peak with black base given is the one where we found HE-4. (<b>B</b>) 12.5% SDS-PAGE of peaks obtained from anion exchanger DEAE sephacel. Lane I: Protein marker. Lane II: Elution profile of First peak in which we obtained HE-4 with other impurities. Lane III, IV and V: Elution profile of second, third and fourh peak respectively. (<b>C</b>) Elution profile of Sephadex G-75. The peaks were obtained as a function of X axis as elution volume in mL and Yaxis is mA at 280 nm. Buffer used for elution is 50 mM Tris-HCl pH 8.0 and 0.2 M NaCl. (<b>D</b>) 12% SDS-PAGE of the third peak of elution profile of sephadex G-75. Lane I: Protein Marker. Lane II: Elution of the third peak in non-reducing condition and Lane III: Elution profile of a third peak in reducing conditions. (<b>E</b>) Immunodetection of HE-4 in crude seminal plasma and of the third peak of elution profile of sephadex G-75. Lane I: Crude seminal plasma. Lane II: Purified protein (IIIrd peak of sephadex G-75). (<b>F</b>) The standard curve for Sephadex G-75 and lines drawn for the position of HE-4 in the standard curve.</p

14% SDS-PAGE to resolve HE-4 and pepsin, papain incubated alone or together for the same duration.

<p>(<b>A</b>) 14% SDS-PAGE (silver stained) Lane1: Molecular weight marker. Lane2: Fresh HE-4. Lane3: HE-4 after 1 hr incubation. Lane 4: Fresh papain. Lane5: papain after 1 hr incubation. Lane 6- Lane 10 papain and HE-4 in 1∶1–1∶5 (10 µg∶10 µg-10 µg∶50 µg) ratio after 1 hr incubation (in 50 mM tris-HCl buffer, pH 8.5). (<b>B</b>) 14% SDS-PAGE (silver stained) Lane1: Molecular weight marker. Lane2: Fresh HE-4 (10 µg). Lane3: HE-4 after 1 hr incubation (10 µg). Lane 4: Fresh pepsin (10 µg). Lane5: pepsin (10 µg) after 1 hr incubation. Lane 6- Lane 10 pepsin and HE-4 in 1∶1–1∶5 ratio (10 µg∶10 µg–10 µg∶50 µg) after 1 hr incubation (in 50 mM sodium acetate buffer, pH 5.0). (<b>C</b>) Immunodetection of HE-4 after incubating HE-4 alone and with pepsin and papain for the same duration. Lane1: HE-4 (10 µg) after 1 hr incubation. Lane 2: HE-4 (10 µg) after 1 hr incubation with papain (10 µg) and Lane3: HE-4 (10 µg) 1 hr incubation with pepsin (10 µg).</p

N‑Glycoproteomics of Human Seminal Plasma Glycoproteins

Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample

Multiple sequence alignment of HE-4 with other WAP protein and HE-4 of different species.

<p>At the end of each line of amino acid sequence, amino acid residue numbers are given and after that % homology is written. (<b>A</b>) Sequence alignment of HE-4 WAP 1 domain with other antiproteases like elafin and WAP-2 domain of SLPI. (<b>B</b>) Sequence alignment of HE-4 WAP2 domain with other antiproteases like elafin and SLPI. (<b>C</b>) Multiple sequence alignment of HE-4 protein of human with HE-4 protein of different species.</p