The TEAD transcription factors are the final effectors of the Hippo pathway, and to exert their transcriptional activity they need to interact with other proteins. The three paralogous vestigial-like proteins VGLL1, VGLL2 and VGLL3 bind to TEAD via a conserved short linear sequence, the Tondu motif. The TEAD-binding domain of human VGLL2 contains in addition an Ω-loop, which is also present in Vg (vestigial) from arthropods and the YAP proteins, another family of TEAD interactors. In this report, using the available structural data, we study the amino acid sequence of the TEAD-binding domain of more than 2400 putative VGLL proteins from vertebrates. This analysis shows a strong link between sequence conservation and functional role for the residues from the Tondu motif. It also reveals that one protein sequence containing both a Tondu motif and an Ω-loop is present in most (if not all) vertebrate species. This suggests that there is a selective pressure to keep a VGLL paralog with a functional Ω-loop in vertebrates. Finally, this study identifies, particularly in mammals, variants of VGLL2 and VGLL3 with an altered TEAD-binding domain suggesting that they may have a different biological function than their homologs.

The GH78 α-L-rhamnosidase from Aspergillus tubingensis (AT-Rha) was proved to be a new clade of Aspergillus α-L-rhamnosidases in the previous study. A putative α-L-rhamnosidase from A. kawachii IFO 4308 (AK-Rha) has 92 % identity in amino acid sequence with AT-Rha. In this study, AK-Rha was expressed in P. pastoris and characterized. Similar to AT-rRha, the recombinant AK-Rha (AK-rRha) showed a narrow substrate specificity to naringin. Interestingly, the enzyme activity of AK-rRha was 0.816 U/mg toward naringin, significantly lower than 125.142 U/mg of AT-rRha. Their large differences in catalytic efficiency was mainly due to their differences in kcat values between AK-rRha (0.67 s-1) and AT-rRha (4.89 × 104 s-1). The molecular dynamics simulation exhibited that the overall conformation of AK-Rha was rigid and that of AT-Rha was flexible; the Loop Y-L located above the catalytic domain formed different steric hindrances to naringin, and interacted with the flavonoid matrices at different strengths. The polar solvation energy analysis implied that the glycosidic bond was more easily hydrolysed in AT-Rha. The comparative study verified that the main feature of AK-Rha and AT-Rha represented Aspergillus α-L-rhamnosidase was the narrow substrate specificity toward naringin, and provided an insight of the relationships between their catalytic abilities and structures.

NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/β/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.

Methyl farnesoate epoxidase (MFE) is a gene encoding an enzyme related to the last step of juvenile hormone biosynthesis. Mn-MFE cDNA has a total length of 1695 bp and an open reading frame (ORF) length of 1482 bp, encoding 493 amino acids. Sequence analysis showed that its amino acid sequence has a PPGP hinge, an FGCG structural domain, and other structural domains specific to the P450 family of enzymes. Mn-MFE was most highly expressed in the hepatopancreas, followed by the ovary and gill, weakly expressed in heart and muscle tissue, and barely expressed in the eyestalk and cranial ganglion. Mn-MFE expression remained stable during the larval period, during which it mainly played a critical role in gonadal differentiation. Expression in the ovary was positively correlated and expression in the hepatopancreas was negatively correlated with ovarian development. In situ hybridization (ISH) showed that the signal was expressed in the oocyte, nucleus, cell membrane and follicular cells, and the intensity of expression was strongest at stage O-IV. The knockdown of Mn-MFE resulted in a significantly lower gonadosomatic index and percentage of ovaries past stage O-III compared to the control group. However, no differences were found in the cumulative frequency of molting between the experimental and control groups. Moreover, the analysis of ovarian tissue sections at the end of the experiment showed differences between groups in development speed but not in subcellular structure. These results demonstrate that Mn-MFE promotes the ovarian development of Macrobrachium nipponense adults but has no effect on molting.

The adaptation of amphibians to diverse environments is closely related to the characteristics of their skin. The complex glandular system of frog skin plays a pivotal role in enabling these animals to thrive in both aquatic and terrestrial habitats and consists of crucial functions such as respiration and water balance as well as serving as a defensive barrier due to the secretion of bioactive compounds. We herein report the first investigation on the skin secretion of Odontophrynus americanus, as a potential source of bioactive peptides and also as an indicator of its evolutionary adaptations to changing environments. Americanin-1 was isolated and identified as a neutral peptide exhibiting moderate antibacterial activity against E. coli. Its amphipathic sequence including 19 amino acids and showing a propensity for α-helix structure is discussed. Comparisons of the histomorphology of the skin of O. americanus with other previously documented species within the same genus revealed distinctive features in the Patagonian specimen, differing from conspecifics from other Argentine provinces. The presence of the Eberth-Katschenko layer, a prevalence of iridophores, and the existence of glycoconjugates in its serous glands suggest that the integument is adapted to retain skin moisture. This adaptation is consistent with the prevailing aridity of its native habitat.

The human cationic antimicrobial protein (hCAP) corresponding to the overlapping sequences of 151-162 of hCAP named KR-12 peptide is the smallest portion of the only type of human Cathelicidin, which has been shown to be modifiable into a more effective antimicrobial. In this study, an in silico analysis, supported by potentiometric titration and isothermal titration calorimetry techniques, was performed to identify potential Cu(II) binding sites of KR-12. The analysis of the presented data at the given theoretical level (GFN2-xTB/ALPB) revealed which peptide chain fragments are involved in the most favourable KR-12-Cu(II) binding mode. Based on a quantum chemical approach, the most favourable coordination modes of Cu(II) to peptides are proposed together with the discussion of the chemical nature of the interactions. The presented results demonstrated that KR-12 interacts with metal ions mostly via the main chain\'s oxygen atoms; however, the two types of amino acids that are expected to be vital for the interaction of Cu(II) are D (aspartic acid) and R29 (arginine). It was demonstrated that in order to explain the complexity of the interaction process in peptide-metal ion systems, the use of theoretical methods is sometimes necessary to explain the details of the experimental results and provide an in-depth understanding of these dynamic systems.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes.

Antimicrobial peptides (AMPs) are of growing interest as potential candidates that may offer more resilience against antimicrobial resistance than traditional antibiotic agents. In this article, we perform the first in silico study of the synthetic ß sheet-forming AMP GL13K. Through atomistic simulations of single and multi-peptide systems under different conditions, we are able to shine a light on the short timescales of early aggregation. We find that isolated peptide conformations are primarily dictated by sequence rather than charge, whereas changing charge has a significant impact on the conformational free energy landscape of multi-peptide systems. We demonstrate that the loss of charge-charge repulsion is a sufficient minimal model for experimentally observed aggregation. Overall, our work explores the molecular biophysical underpinnings of the first stages of aggregation of a unique AMP, laying necessary groundwork for its further development as an antibiotic candidate.

Intrinsically disordered proteins (IDPs) are known for their random structural changes throughout their sequence based on the environment. The mechanism underlying these structural changes is difficult to explain. All biological processes are known to follow the direction through which they act. A study of the correlated motion can help to understand the direction of the change. Herein, we introduced the multivariate statistical analysis (MSA) technique to study the correlated motion of the peptide. The correlated motion of the sheep prion peptide was studied with the change in the temperature and solvent. These techniques helped to identify the contributing residual motions that helped to form the different secondary structures of the protein and also the triggering factors that drive these sorts of residual motions. The structural details match the experimentally reported data. It was found that the direction of the change of the secondary structure for this peptide shifted from the C-terminal to the N-terminal with an increase in the temperature. It was found that the involvement of the hydrophobic residues present at the C-terminal and the middle residues (residues 12-17) is responsible for forming a β-sheet at the normal temperature. Hydration water was found to play an important role in this change. Insights gained from this study can be used to design strategies for desirable structural changes in the IDPs.