A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome

BACKGROUND: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done. RESULTS: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice. CONCLUSIONS: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study

Conformation and activity of δ-lysin and its analogs

δ-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, δ-lysin does not exhibit antibacterial activity. We have synthesized δ-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of δ-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike δ-lysin, did not self-associate to adopt α-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on δ-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities

Tyrosine phosphoproteome of hamster spermatozoa: role of glycerol-3-phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine-phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol-3-phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation

Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death

The gramicidin pore: selective tryptophan replacement with aspartic acid

The chemoselective transformation of Trp9 and Trp11, located at the hydrophobic core of the antiparallel, double helical pore of O-acetyl gramicidin A(HCO-ValGlyAlaLeuoAlaValoValValo TrpLeuo TrpLeuoTrpLeuoTrpNHCH2CH2OAc; o = D isomer) to Asp9 gramicidin and Asp9 Asp11 gramicidin has been demonstrated by oxidation with 0.25 eq. of in situ generated Ru(VIII), HPLC isolation, amino acid analysis and peptide sequencing

Ubiquitous lens α-, β-, and γ-crystallins accumulate in anuran cornea as corneal crystallins

Corneal epithelium is known to have high levels of some metabolic enzymes such as aldehyde dehydrogenase in mammals, gelsolin in zebrafish, and α-enolase in several species. Analogous to lens crystallins, these enzymes and proteins are referred to as corneal crystallins, although their precise function is not established in any species. Although it is known that after lentectomy, the outer cornea undergoes transdifferentiation to regenerate a lens only in anuran amphibians, major proteins expressed in an anuran cornea have not been identified. This study therefore aimed to identify the major corneal proteins in the Indian toad (Bufo melanostictus) and the Indian frog (Rana tigrina). Soluble proteins of toad and frog corneas were resolved on two-dimensional gels and identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight and electrospray ionization quadrupole time-of-flight. We report that anuran cornea is made up of the full complement of ubiquitous lens α-, β-, and γ-crystallins, mainly localized in the corneal epithelium. In addition, some taxon-specific lens crystallins and novel proteins, such as α- or β-enolase/γ-crystallin, were also identified. Our data present a unique case of the anuran cornea where the same crystallins are used in the lens and in the cornea, thus supporting the earlier idea that crystallins are essential for the visual functions of the cornea as they perform for the lens. High levels of lens α-, β-, and γ-crystallins have not been reported in the cornea of any species studied so far and may offer a possible explanation for their inability to regenerate a lens after lentectomy. Our data that anuran cornea has an abundant quantity of almost all the lens crystallins are consistent with its ability to form a lens, and this connection is worthy of further studies

Protein sub networks of fluvastatin mediated differentially regulated proteins.

<p>Protein-protein physical/functional interaction global complex network (A) and sub network (B) generated by Ingenuity Pathway Analysis tool. Grey filled boxes are the differentially expressed proteins. Only significant sub networks are represented.</p

Caspase-3 inhibitor but not proteasomal inhibitor alters fluvastatin-mediated regulation of vimentin levels in MDA-MB-231 cells.

<p>A, MDA-MB-231 cells were treated with various concentrations of fluvastatin (0–20 µM) for a period of 24 h and at the end of the treatments trypsin-like and chymotrypsin-like activities of the 26S proteasome were measured using respective fluorogenic substrates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108890#s2" target="_blank">materials and methods</a> section. B, is same as A except that cells were treated with fluvastatin (10 µM) for various time points (0–24 h). C, MDA-MB-231 cells were treated in the presence or absence of MG-132 (1 µM) for a period of 24 h and at the end of the treatments, vimentin and β-catenin protein levels were measured by Western blot analysis. D, MDA-MB-231 cells were pre-treated with caspase-3 inhibitor (50 µM) for a period of 6 h in the presence or absence of fluvastatin (10 µM) for a period of 24 h and vimentin protein levels were measured by Western analysis. Data represented are Mean±SD from at least three independent experiments. *, significantly different compared to untreated conditions; #, significantly different compared to MG-132 and fluva+MG-132 condition; **, significantly different compared to fluvastatin alone treated condition. Statistical significance was tested at P<0.05 level by two-tailed, unpaired, Student's t-test.</p

Impact of Storage Conditions on the Breast Milk Peptidome

Human donor milk (HDM) provides appropriate nutrition and offers protective functionsin preterm infants. The aim of the study is to examine the impact of different storage conditions onthe stability of the human breast milk peptidome. HDM was directly frozen at−80◦C or stored at−20◦C (120 h), 4◦C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiledby mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storagetemperature and time and varied for different peptides. The highest impact was observed whensamples were stored at RT. Smaller but significant effects were still observed in samples stored at4◦C, while samples showed highest similarity to those immediately frozen at−80◦C when storedat−20◦C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombinand other proteases cleaving proteins at lysine/arginine. The results point to an ongoing proteindegradation/peptide production by milk-derived proteases. They underline the need for immediatefreezing of HDM at−20◦C or−80◦C to prevent degradation of peptides and enable reproducibleinvestigation of prospectively collected samples

Fluvastatin induces cell death in MDA-MB-231 breast cancer cells: Effect of mevalonate.

<p>A, MDA-MB-231 cells were treated with various concentrations of fluvastatin (0–20 µM) for 48 h and B shows cells treated for different time points with 10 µM fluvastatin. At the end of the treatments, cell viability was measured by trypan blue exclusion method. C, MCF-10A cells were treated with fluvastatin (0–20 µM) for a period of 24 and 48 h and cell death was measured as described earlier. D, MDA-MB-231 cells were treated with fluvatstatin (10 µM) in presence or absence of mevalonate (25 µM) for a period of 48 h and cell viability was measured by trypan blue exclusion assay. Data represented is Mean±SD from at least three independent experiments. *, significantly different compared to untreated conditions; #, significantly different compared to mevalonate and fluva+mev condition. Statistical significance was tested at P<0.05 level by two-tailed, unpaired, Student's t-test.</p