Optimization of Small Peptide Feed from Milk Thistle Residue by Synergistic Fermentation of Multiple Strains and Proteases

In order to improve the utilization rate of the milk thistle residue, this study used the synergistic fermentation of multiple strains and proteases to increase the small peptide content of the fermented feed produced by the milk thistle residue. Taking the small peptide content of the milk thistle residue fermented feed as an indicator, the optimal fermentation process was obtained by single-factor optimization experiments and the response surface methodology. The optimal fermentation process was as follows: fermentation time of 7 days, inoculum size of 15%, inoculation ratio of aerobic strains: anaerobic strains = 1: 2, solid-state fermentation water content of 66%, fermentation temperature of 36℃, and amount of protease was 0.25% acid protease+0.25% bromelain. Under the above process, the small peptide content of the fermented feed from milk thistle residue was greatly improved to 57.86%. These results inferred that the added proteases were beneficial to the growth of fermentative microorganisms, the secretion of protease and the increase of the small peptide content.

Molecular Dynamics Simulations of HLA-CW4-Β2M-KIR2DL1 Protein and Homology Modeling of a Complex Associated with Psoriasis Disease (HLA-CW6-Β 2M-KIR2DS1)

Molecular dynamics (MD) simulation was used to study the interactions of two immune proteins of HLA-Cw4-β2m-KIR2DL1 complex with small peptide QYDDAVYKL (nine amino acids) in an aqueous solution. This study aims to gain a detailed information about the conformational changes and the dynamics of the complex. The right parameters and force field for performing the MD simulations that was needed to calibrate the complex structure were determined. The non-bonded interactions (Electrostatic and van der Waals contributions), H-bond formation, and salt bridges between the ligand HLA-Cw4 and the receptor KIR2DL1 were estimated using the obtained MD trajectories. The buried surface area due to binding was calculated to get insight into the causes of specificity of receptor to ligand and explains mutations experiment. The study concluded that β2-microglobulin, one part of the complex, is not directly interacting with the peptide at the groove; therefore, it could be neglected from simulation. Our results showed that β2-microglobulin does not have any significant effect on the dynamics of the 3D-structure of the complex. This project will help in understanding to optimize candidate drug design, a small peptide that disrupts the interaction, for the optimal biological effect.

KSHV transactivator-derived small peptide traps coactivators to attenuate MYC and inhibits leukemia and lymphoma cell growth

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

OaAEP1-mediated PNA-protein conjugation enables erasable imaging of membrane protein

Methods to efficiently and site-specifically conjugate proteins to nucleic acids could enable exciting application in bioanalytics and biotechnology. Here, we report the use of the strict protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation requires only a short N-terminal GL dipeptide in target protein and a C-terminal NGL tripeptide in PNA. We demonstrate the versatility of this approach by conjugating three different types of proteins with a PNA strand. The biostable PNA strand then serves as a generic landing platform for nucleic acid hybridization. Lastly, we show the erasable imaging of EGFR on HEK293 cell membrane through toehold-mediated strand displacement. This work provides a controlled tool for precise conjugation of proteins with nucleic acids through an extremely small peptide linker and facilitates further study of membrane proteins.

Serum Prokineticin-2 in Prepubertal and Adult Klinefelter Individuals

The prokineticin-2 (PROK2) is a small peptide belonging to the prokineticin family. In humans and rodents this chemokine is primarily involved in the control of central and peripheral reproductive processes. Klinefelter's syndrome (KS) is the first cause of male genetic infertility, due to an extra X chromosome, which may occur with a classical karyotype (47, XXY) or mosaic forms (46, XY/47, XXY). In affected subjects, pubertal maturation usually begins at an adequate chronological age, but when development is almost complete, they display a primary gonadal failure, with early spermatogenesis damage, and later onset of testosterone insufficiency. Thus, the main aim of the present study was to investigate the serum levels of PROK2 in prepubertal and adult KS patients, comparing them with healthy subjects. We showed for the first time the presence of PROK2 in the children serum but with significant changes in KS individuals. Indeed, compared to healthy subjects characterized by PROK2 serum elevation during the growth, KS individuals showed constant serum levels during the sexual maturation phase (higher during the prepubertal phase but lower during the adult age). In conclusion, these data indicate that in KS individuals PROK2 may be considered a biomarker for investigating the SK infertility process.

KSHV transactivator-derived small peptide traps coactivators to attenuate MYC function and kills leukemia and lymphoma

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we identified a small peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular c-MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (​SLAM seq) with target-transcriptional analyses further confirmed that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may have significant potential as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists

EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.