Matrix is everywhere: extracellular DNA is a link between biofilm and mineralization in Bacillus cereus planktonic lifestyle.

To date, the mechanisms of biomineralization induced by bacterial cells in the context of biofilm formation remain the subject of intensive studies. In this study, we analyzed the influence of the medium components on the induction of CaCO3 precipitation by the Bacillus cereus cells and composition of the extracellular matrix (ECM) formed in the submerged culture. While the accumulation of extracellular polysaccharides and amyloids appeared to be independent of the presence of calcium and urea during the growth, the accumulation of extracellular DNA (eDNA), as well as precipitation of calcium carbonate, required the presence of both ingredients in the medium. Removal of eDNA, which was sensitive to treatment by DNase, did not affect other matrix components but resulted in disruption of cell network formation and a sixfold decrease in the precipitate yield. An experiment with a cell-free system confirmed the acceleration of mineral formation after the addition of exogenous salmon sperm DNA. The observed pathway for the formation of CaCO3 minerals in B. cereus planktonic culture included a production of exopolysaccharides and negatively charged eDNA lattice promoting local Ca2+ supersaturation, which, together with an increase in the concentration of carbonate ions due to pH rise, resulted in the formation of an insoluble precipitate of calcium carbonate. Precipitation of amorphous CaCO3 on eDNA matrix was followed by crystal formation via the ACC-vaterite-calcite/aragonite pathway and further formation of larger mineral aggregates in complex with extracellular polymeric substances. Taken together, our data showed that DNA in extracellular matrix is an essential factor for triggering the biomineralization in B. cereus planktonic culture.

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry.

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M's 'bait region', and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity.

The influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6-13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures. In this paper, it was shown that PB1(6-13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6-13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6-13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.

A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis.

The fibrillogenesis of a peptide corresponding to residues 35-51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹7) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.

Amyloidogenic peptide homologous to fragment 129-148 of human myocilin.

Myocilin is a protein with a molecular weight near 50 kDa. It is expressed in almost all organs and tissues. We showed that the peptide DQLETQTRELETAYSNLLRD corresponding to N-terminal Leucine zipper motif (LZM) of the protein is able to form amyloid-like fibrils. The possible role of this motif in myocilin aggregation is discussed.

Structural Features of the Peptide Homologous to 6-25 Fragment of Influenza A PB1 Protein.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.

Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1.

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.