Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Multiple regions of junctin drive interaction with calsequestrin-1 and localization at triads in skeletal muscle

Junctin is a transmembrane protein of striated muscles, localized at the junctional sarcoplasmic reticulum (j-SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor. Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids. However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins co-localize at triads, where they assemble with other j-SR proteins. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs able to bind calsequestrin. In addition, stretches of charged amino acids downstream these motifs were found to be also able to bind calsequestrin and the ryanodine receptor. Deletion of even one of these regions impaired the ability of junctin to localize at the j-SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

Predicting antibiotic resistance in complex protein targets using alchemical free energy methods

Multi-drug resistant Mycobacterium tuberculosis requires a complex antibiotic treatment program and poses a major threat to tuberculosis (TB) treatment outcomes. Resistance is mostly conferred by chromosomal single nucleotide polymorphisms, many of which are well characterized and catalogued. However, not all mutations have been mapped and novel mutations can emerge. Methods able to quickly predict the effects of such mutations are needed to complement the existing catalogues, thereby permitting the prescription of effective treatment for patients and preventing the further spread of resistant strains. Relative binding free energy (RBFE) calculations can rapidly predict the effects of mutations, but this approach has not been tested on large, complex proteins. We use RBFE calculations to predict the effects of seven M. tuberculosis RNA polymerase mutations on rifampicin susceptibility and five M. tuberculosis DNA gyrase mutations on moxifloxacin susceptibility. These mutations encompass a range of amino acid substitutions with known effects and include large steric perturbations and charged moieties. We find that moderate numbers (n=3-15) of short RBFE calculations can predict resistance in cases where the mutation results in a large change in the binding free energy, but that the method lacks discrimination in cases with either a small change in energy or that involve charged amino acids, due to the associated large magnitude of error. We investigate how this error may be decreased by analyzing the sources of error and the distributions of repeated measurements from the different components of the RBFE calculations.

PHYSICO-CHEMICAL BASES OF THE USE OF DIELECTRIC SEPARATION TO IMPROVE THE QUALITY OF PROCESSING OF SEEDS OF SPICY-AROMATIC PLANTS

Исследованы биологические особенности (морфология поверхности, а также качественный и количественный состав белков) семян (плодов) пряно-ароматических растений: кориандра (Coriandrum sativumL.), тмина (Carum carvi L.) и укропа (Anethum graveolens L.). Методами сканирующей электронной микроскопии установлена нерегулярная поверхность семян кориандра, тмина и укропа. На их поверхности идентифицировано большое количество бороздок, углублений, бугорков. Поверхность шероховатая и содержит много волосков. Особенно много волосков имеется на поверхности семян тмина. Определено, что в 100 г семян кориандра, тмина и укропа содержится: общего белка – 12,37; 19,77 и 15,98 г соответственно, свободных аминокислот – 1,42; 18,46 и 8,39 г соответственно. Сумма положительно заряженных незаменимых аминокислот (аргинина, гистидина и лизина) в 100 г кориандра, тмина и укропа составляет соответственно 0,149; 2,833 и 2,621 г, сумма отрицательно заряженных аминокислот (аспарагиновой и глутаминовой) – 0,71; 5,253 и 0,633 г соответственно. Результаты исследований будут способствовать совершенствованию технологии глубокой переработки растительного сырья для получения семян высокого качества с заданными свойствами путем отделения примесей органической и неорганической природы. The biological features (surface morphology, as well as the qualitative and quantitative composition of proteins) of seeds of spicy-aromatic plants: coriander (Coriandrum sativum L.), cumin (Carum carvi L.) and dill (Anethum graveolens L.) were studied. The irregular surface of coriander, cumin and dill seeds was established by scanning electron microscopy. A large number of grooves, depressions, and bumps have been identified on their surface. The surface is rough and contains a lot of hairs. There are especially many hairs on the surface of cumin seeds. It was determined that 100 g of coriander, cumin and dill seeds contain: total protein – 12,37; 19,77 and 15,98 g respectively, free amino acids – 1,42; 18,46 and 8,39 g respectively. The sum of positively charged essential amino acids (arginine, histidine and lysine) in 100 g of coriander, cumin and dill is 0,149, 2,833 and 2,621 g respectively, the sum of negatively charged amino acids (aspartic and glutamic) is 0,71; 5,253 and 0,633 g respectively. The results of the research will contribute to the improvement of the technology of deep processing of plant raw materials to obtain high-quality seeds with desired properties by separating impurities of organic and inorganic nature.

Tracking the movement of discrete gating charges in a voltage-gated potassium channel

Positively-charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1, R2) in the Shaker potassium channel voltage sensor using a fluorescent positively-charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage sensitive phosphatase (CiVSP) (Murata et al., 2005a).

Amino Acid Dependent Performance of Modified Chitosan Against Bacteria and Red Blood Cell

In order to combat antibiotic resistance, the development of new antibacterial agents is essential. In this study, we prepared four types of amino acid modified chitosan (CS-AA). Compared with chitosan modified with hydrophobic amino acids, the chitosan modified with positively charged amino acids showed higher antibacterial efficiency against Escherichia coli (E. coli) under similar grafting rate. CS-AA achieves antibacterial properties mainly by destroying the integrity of bacterial cell membranes. All the four types of CS-AA show low toxicity towards red blood cells. This work indicates that positively charged groups are more important than hydrophobic groups in the design of chitosan-based antibacterial agents, and provides helpful information for the molecular design of effective antibacterial agents.

Altering Amino Acid Profile in Catharanthus Roseus (L.) G. Don Using Potassium and Ascorbic Acid Treatments

Background: Catharanthus roseus (L.) G. Don is the main source of alkaloids anticancer drugs. Alkaloids are derived from amino acids and can lead to changes in these valuable compounds. Objective: This experiment evaluated the variation of amino acids under potassium and ascorbic acid treatments. Methods: Different concentrations (1.5, 3.16, 15 and 30 mM) and forms (K2SO4 and KNO3) of potassium (K+) were added to plants via Hoagland’s nutrient solution. Ascorbic acid (AsA) (750 mg L-1) was sprayed on the leaves surfaces on days 68 and 78. Amino acids were extracted from 90-day-old plant leaves, and different amino acids were determined by High-Performance Liquid Chromatography with fluorescence detection. Results: Amino acids increased in K+ deficiency (1.5 mM), but changes in negatively charged amino acids were lower. In contrast, N-rich amino acids showed the biggest change. In excessive K+, branched-chain and aromatic amino acids decreased, while the least amount of the other amino acids was observed in the plants treated with optimum K+. Conclusion: The exterior of AsA and excessive K+ cause branched-chain and aromatic amino acids, aspartic acid and glutamic acid to decrease.

Molecular coevolution of nuclear and nucleolar localization signals inside basic domain of HIV-1 Tat

During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, to reduce protein size, influencing the origin and evolution of NLSs and NoLSs in viruses. IMPORTANCE Here, we investigated the molecular mechanism of NLS and NoLS integration into the basic domain of HIV-1 Tat ( 49 RKKRRQRRR 57 ), and found that these two supplementary functions (i.e., function of NLS and NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

Lysine Residues in the MK-Rich Region Are Not Required for Binding of the PbsP Protein From Group B Streptococci to Plasminogen

Binding to plasminogen (Plg) enables bacteria to associate with and invade host tissues. The cell wall protein PbsP significantly contributes to the ability of group B streptococci, a frequent cause of invasive infection, to bind Plg. Here we sought to identify the molecular regions involved in the interactions between Plg and PbsP. The K4 Kringle domain of the Plg molecule was required for binding of Plg to whole PbsP and to a PbsP fragment encompassing a region rich in methionine and lysine (MK-rich domain). These interactions were inhibited by free L-lysine, indicating the involvement of lysine binding sites in the Plg molecule. However, mutation to alanine of all lysine residues in the MK-rich domain did not decrease its ability to bind Plg. Collectively, our data identify a novel bacterial sequence that can interact with lysine binding sites in the Plg molecule. Notably, such binding did not require the presence of lysine or other positively charged amino acids in the bacterial receptor. These data may be useful for developing alternative therapeutic strategies aimed at blocking interactions between group B streptococci and Plg.