Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.     

Phallus impudicus loaded with γ-Fe2O3 as solid phase bioextractor for the preconcentrations of Zn(II) and Cr(III) from water and food samples

We investigated the application of fungus Phallus impudicus loaded γ-Fe₂O₃ nanoparticles as a biosorbent for magnetic solid phase extractions of trace levels of Zn(II) and Cr(III) ions from natural samples before their measurements by inductively coupled plasma optical emission spectrometry. The characterization of magnetized P. impudicus was performed using the scanning electron microscope, the energy dispersive X-ray and Fourier transform infrared spectroscopy. Important experimental factors were investigated. The experimental results fitted well to the Langmuir adsorption model and pseudo-second order kinetic model. Limit of detections of targeted ions by magnetic solid phase extraction method based on use of P. impudicus were found as 10.5 ngL⁻¹ and 12.6 ngL⁻¹ respectively for Cr(III) and Zn(II). The sorption capacities of the biosorbent were 22.8 mgg⁻¹ for Cr(III) and 25.6 mgg⁻¹ for Zn(II). The preconcentration factors were achieved as 100 for both of ions. RSDs for inter- and intraday precisions were found as lower than 2.0% and 2.1% respectively for both of targeted ions. The accuracy of the recommended process was tested by recovery measurements on the certificated reference materials and successfully applied for quantification recoveries of Cr(III) and Zn(II) ions from water and food samples.     

A surface molecularly imprinted polymer as chiral stationary phase for chiral separation of 1,1′‐binaphthalene‐2‐naphthol racemates

Acrylamide (AM) was copolymerized with ethylene glycol dimethacrylate (EGDMA) in the presence of (R )‐1,1′‐binaphthalene‐2‐naphthol (BINOL) as the template molecules on the surface of silica gel by a free radical polymerization to produce a chiral stationary phase based on the surface molecularly imprinted polymer (SMIP‐CSP). The SMIP‐CSP showed a much better separation factor (α = 4.28) than the CSP based on the molecularly imprinted polymer (MIP‐CSP) without coating on the silica gel (α = 1.96) during the chiral separation of BINOL enantiomers by high‐performance liquid chromatography. The influence of the pretreatment temperature and the content of the template molecule ((R )‐BINOL) of the SMIP‐CSP, and the mobile phase composition on the separation of the racemic BINOL were systematically investigated.     

An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.     

N-terminal Domain of TDP43 Enhances Liquid-Liquid Phase Separation of Globular Proteins

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43′s NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.     

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.     

An improved breeding method for solitarious locusts

In order to unravel the physiological, endocrine, and behavioral differences between gregarious and solitarious forms of the desert locust, Schistocerca gregaria (Forsk.) (Orthoptera, Acrididae), a constant supply of rather large numbers of solitary individuals has to be guaranteed. This represents a bottleneck, mainly because of the intensity of the labor involved and limited laboratory accommodation. The method we describe here substantially reduces the space and manpower needed. The survival rate we obtained in the solitarised population was relatively high, reaching about 55%. The optimal rearing temperature proved to be 32–36 °C. Cabbage leaves and oat flakes sufficed for feeding all year round. Special racks have been designed that enable high density stacking and easy handling. The solitarisation process was monitored over ten consecutive generations. Changes in morphometrics, eye stripes, color, and behavior were recorded, of which only morphometrics, temperature related development, and mortality are discussed. A shift towards the solitarious phase was recorded, with clear differences between gregarious, 1^st generation and 7^th to 10^th generation solitarious locusts.     

Albumin stabilizes 14,15-leukotriene A4

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.     

Aza-β-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates

Peptidomimetic analogs of the peptide RRASVA, known as the “minimal substrate” of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β³ analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands.     

The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus , designated slp , was sequenced and the recombinant gene product was expressed in Escherichia coli . The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus . However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.