Proteomic profiles and the function of RBP4 in endometrium during embryo implantation phases in pigs.

BACKGROUND: Endometrial receptivity plays a vital role in the success of embryo implantation. However, the temporal proteomic profile of porcine endometrium during embryo implantation is still unclear. RESULTS: In this study, the abundance of proteins in endometrium on days 9, 10, 11, 12, 13, 14, 15 and 18 of pregnancy (D9, 10, 11, 12, 13, 14, 15 and 18) was profiled via iTRAQ technology. The results showed that 25, 55, 103, 91, 100, 120, 149 proteins were up-regulated, and 24, 70, 169, 159, 164, 161, 198 proteins were down-regulated in porcine endometrium on D10, 11, 12, 13, 14, 15 and 18 compared with that on D9, respectively. Among these differentially abundance proteins (DAPs), Multiple Reaction Monitoring (MRM) results indicated that S100A9, S100A12, HRG and IFI6 were differentially abundance in endometrial during embryo implantation period. Bioinformatics analysis showed that the proteins differentially expressed in the 7 comparisons were involved in important processes and pathways related to immunization, endometrial remodeling, which have a vital effect on embryonic implantation. CONCLUSION: Our results reveal that retinol binding protein 4 (RBP4) could regulate the cell proliferation, migration and apoptosis of endometrial epithelial cells and endometrial stromal cells to affect embryo implantation. This research also provides resources for studies of proteins in endometrium during early pregnancy.

Development of the Safe and Broad-Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ-AA Peptide Scaffold.

SARS-CoV-2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (Mpro ) is essential in the replication of SARS-CoV-2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics-constrained α, γ-AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the Mpro of SARS-CoV-2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET-based Mpro enzymatic assay not only for the Mpro of SARS-CoV-2 but also for SARS-CoV and MERS-CoV, along with HCoVs like HCoV-OC43, HCoV-229E, HCoV-NL63 and HKU1. The X-ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS-CoV-2. Overall, the results suggest that α, γ-AA peptide could be a promising molecular scaffold in designing novel Mpro inhibitors of SARS-CoV-2 and other coronaviruses.

Sulfono-γ-AApeptides as Protein Helical Domain Mimetics to Manipulate the Angiogenesis.

Sulfono-γ-AApeptides recently developed in our group have been proven to be a new class of unnatural foldamer with well-defined helical structure and have been demonstrated to mimic protein helical domains and disrupt biomedically relevant protein-protein interactions (PPIs). Based on our design concept in a recent report, we discovered two similar sulfono-γ-AApeptides V2 and V3 which were designed to mimic the VEGF N-terminal helix α1 known to directly interact with VEGFRs. Interestingly, V2 was shown to possess the pro-angiogenic effect, whereas V3 was proved to be a potent inhibitor for angiogenesis. We speculate that the distinct angiogenesis signaling was due to the selective binding of the two molecules to VEGFR1 and VEGFR2, respectively. Together with their remarkable resistance to proteolytic degradation, relatively small sizes, and amenability to modification with diverse functional groups, V2 and V3 could serve as lead molecules for the development of potential therapeutic agents and molecular probes. These findings highlight sulfono-γ-AApeptides as an alternative paradigm to mimic the α-helical domain to modulate a wide variety of PPIs in the future.

Sulfono-γ-AApeptides as Helical Mimetics: Crystal Structures and Applications.

Foldamers have defined and predictable structures, improved resistance to proteolytic degradation, enhanced chemical diversity, and are versatile in their mimicry of biological molecules, making them promising candidates in biomedical and material applications. However, as natural macromolecules exhibit endless folding structures and functions, the exploration of the applications of foldamers remains crucial. As such, it is imperative to continue to discover unnatural foldameric architectures with new frameworks and molecular scaffolds. To this end, we recently developed a new class of peptidomimetics termed ″γ-AApeptides", oligomers of γ-substituted-N-acylated-N-aminoethyl amino acids, which are inspired by the chiral peptide nucleic acid backbone. To date γ-AApeptides have been shown to be resistant to proteolytic degradation and possess limitless potential to introduce chemically diverse functional groups, demonstrating promise in biomedical and material sciences. However, the structures of γ-AApeptides were initially unknown, rendering their rational design for the mimicry of a protein helical domain impossible in the beginning, which limited their potential development. To our delight, in the past few years, we have obtained a series of crystal structures of helical sulfono-γ-AApeptides, a subclass of γ-AApeptides. The single-crystal X-ray crystallography indicates that sulfono-γ-AApeptides fold into unprecedented and well-defined helices with unique helical parameters. On the basis of the well-established size, shape, and folding conformation, the design of sulfono-γ-AApeptide-based foldamers opens a new avenue for the development of alternative unnatural peptidomimetics for their potential applications in chemistry, biology, medicine, materials science, and so on.In this Account, we will outline our journey on sulfono-γ-AApeptides and their application as helical mimetics. We will first briefly introduce the design and synthetic strategy of sulfono-γ-AApeptides and then describe the crystal structures of helical sulfono-γ-AApeptides, including left-handed homogeneous sulfono-γ-AApeptides, right-handed 1:1 α/sulfono-γ-AA peptide hybrids, and right-handed 2:1 α/sulfono-γ-AA peptide hybrids. After that, we will illustrate the potential of helical sulfono-γ-AApeptides for biological applications such as the disruption of medicinally relevant protein-protein interactions (PPIs) of BCL9-ß-catenin and p53-MDM2/MDMX as well as the mimicry of glucagon-like peptide 1 (GLP-1). In addition, we also exemplify their potential application in material science. We expect that this Account will shed light on the structure-based design and function of helical sulfono-γ-AApeptides, which can provide a new and alternative way to explore and generate novel foldamers with distinctive structural and functional properties.

mRNA/lncRNA expression patterns and the function of fibrinogen-like protein 2 in Meishan pig endometrium during the preimplantation phases.

Embryonic implantation involves a complex and well-coordinated interaction between the developing conceptus and maternal uterus, and the preimplantation period has a major impact on litter size in pigs. The present study aimed to investigate the vital messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) that regulate preimplantation in Meishan pigs. The enriched Gene Ontology terms were all related to "binding." Furthermore, "ECM-receptor interaction" was predicted as an important pathway that regulated the success of implantation. We speculated that the differentially expressed mRNAs S100A9, ANXA8, MUC16, and FGL2 and the differentially expressed lncRNAs TCONS_11206566, TCONS_09904861, and TCONS_1252933 may play vital roles in the process of implantation. Furthermore, this study verified that FGL2 was highly expressed on Day 12 of pregnancy, and we also investigated the function of FGL2 during preimplantation in vivo. In conclusion, this study provides useful information for further analyses of the molecular mechanisms of implantation in Chinese domestic pigs.

Annexin A8 (ANXA8) regulates proliferation of porcine endometrial cells via Akt signalling pathway.

Annexin A8 (ANXA8) gene, a member of the annexin family, encodes an anticoagulant protein involved in blood coagulation cascade and acts as an indirect inhibitor of the thromboplastin-specific complex. However, little is known about the function of ANXA8 in porcine endometrial cells so far. Here, ANXA8 mRNA was found to be abundant in porcine endometrium on days 11-13 of pregnancy. Real-time RT-PCR analysis indicated that the mRNA expression of the leukaemia inhibitory factor (LIF) and the epidermal growth factor (EGF) was upregulated by ANXA8 in porcine endometrial cells. Immunofluorescence technology and cell cycle analysis revealed that ANXA8 promoted the proliferation of endometrial cells, as evidenced by the abundant proliferating cell nuclear antigen (PCNA) expression and an increase in the S phase. Western blot analysis results indicated that ANXA8 activated the phosphorylation of the target protein kinase B (Akt) protein. Immunofluorescence technology results showed that the PCNA protein had no significant change in porcine endometrial cells with both ANXA8 overexpression and the addition of Akt inhibitor. Furthermore, the number of implantation sites was significantly reduced by injection of mus-siRNA-ANXA8 into the uterine horn of mice. Collectively, these results suggest that ANXA8 promotes the proliferation of endometrial cells through the Akt signalling pathway.

Identification of non-coding and coding RNAs in porcine endometrium.

One of the most critical periods of embryonic loss in pig is day 12 of pregnancy, when implantation begins. Here, we analyzed the gene expression on day 12 of pregnancy and non-pregnancy in the porcine endometrium using RNA sequencing (RNA-seq). 237 mRNAs, 34 lncRNAs and 1 miRNA were significantly differentially expressed between the two groups. Further functional analyses were conducted to identify these differentially expressed transcripts. The results demonstrated that they participate in various biological processes, such as cell adhesion, binding, nucleic and metabolic processes. In addition, our results showed that the differentially expressed genes (IL1R, FGF9, DUPS10, DUPS4, CD14 and MAP4K4) in MAPK pathway, and lncRNAs of XLOC_2604764 and XLOC_2604756 may play a vital role in regulating embryo implantation. Besides, we investigated the lncRNA-ssc-miR-132-mRNA interactions, aiming to explain the regulatory networks of coding and non-coding genes that contributes to the establishment of the maternal pregnancy.

Rational Design of Sulfonyl-γ-AApeptides as Highly Potent HIV-1 Fusion Inhibitors with Broad-Spectrum Activity.

The HIV-1 epidemic has significant social and economic implications for public health. Developing new antivirus drugs to eradicate drug resistance is still urgently needed. Herein, we demonstrated that sulfonyl-γ-AApeptides could be designed to mimic MTSC22EK, one potent HIV fusion inhibitor derived from CHR. The best two sequences revealed comparable activity to MTSC22EK in an authentic HIV-1 infection assay and exhibited broad-spectrum anti-HIV-1 activity to many HIV-1 clinical isolates. Furthermore, sulfonyl-γ-AApeptides show remarkable resistance to proteolysis and favorable permeability in PAMPA-GIT and PAMPA-BBB assays, suggesting that both sequences could control HIV-1 within the central nervous system and possess promising oral bioavailability. Mechanistic investigations suggest that these sulfonyl-γ-AApeptides function by mimicking the CHR of gp41 and tightly bind with NHR, thereby inhibiting the formation of the 6-HB structure necessary for HIV-1 fusion. Overall, our results suggest that sulfonyl-γ-AApeptides represent a new generation of anti-HIV-1 fusion inhibitors. Moreover, this design strategy could be adopted to modulate many of the PPIs.

An HR2-Mimicking Sulfonyl-γ-AApeptide Is a Potent Pan-coronavirus Fusion Inhibitor with Strong Blood-Brain Barrier Permeability, Long Half-Life, and Promising Oral Bioavailability.

Neutralizing antibodies and fusion inhibitory peptides have the potential required to combat the global pandemic caused by SARS-CoV-2 and its variants. However, the lack of oral bioavailability and enzymatic susceptibility limited their application, necessitating the development of novel pan-CoV fusion inhibitors. Herein we report a series of helical peptidomimetics, d-sulfonyl-γ-AApeptides, which effectively mimic the key residues of heptad repeat 2 and interact with heptad repeat 1 in the SARS-CoV-2 S2 subunit, resulting in inhibiting SARS-CoV-2 spike protein-mediated fusion between virus and cell membranes. The leads also displayed broad-spectrum inhibitory activity against a panel of other human CoVs and showed strong potency in vitro and in vivo. Meanwhile, they also demonstrated complete resistance to proteolytic enzymes or human sera and exhibited extremely long half-life in vivo and highly promising oral bioavailability, delineating their potential as pan-CoV fusion inhibitors with the potential to combat SARS-CoV-2 and its variants.

Inhibition of the Ubiquitin Transfer Cascade by a Peptidomimetic Foldamer Mimicking the E2 N-Terminal Helix.

The enzymatic cascades for ubiquitin transfer regulate key cellular processes and are the intense focus of drug development for treating cancer and neurodegenerative diseases. E1 is at the apex of the UB transfer cascade, and molecules inhibiting E1 have shown promising activities against cancer cell proliferation. Compared to small molecules, peptidomimetics have emerged as powerful tools to disrupt the protein-protein interactions (PPI) with less drug resistance and high stability in the cell. Herein, we harnessed the D-sulfono-γ-AA peptide to mimic the N-terminal helix of E2 and thereby inhibit E1-E2 interaction. Two stapled peptidomimetics, M1-S1 and M1-S2, were identified as effective inhibitors to block UB transfer from E1 to E2, as shown by in vitro and cellular assays. Our work suggested that PPIs with the N-terminal helix of E2 at the E1-E2 and E2-E3 interfaces could be a promising target for designing inhibitors against protein ubiquitination pathways in the cell.

Activation of E6AP/UBE3A-Mediated Protein Ubiquitination and Degradation Pathways by a Cyclic γ-AA Peptide.

Manipulating the activities of E3 ubiquitin ligases with chemical ligands holds promise for correcting E3 malfunctions and repurposing the E3s for induced protein degradation in the cell. Herein, we report an alternative strategy to proteolysis-targeting chimeras (PROTACs) and molecular glues to induce protein degradation by constructing and screening a γ-AA peptide library for cyclic peptidomimetics binding to the HECT domain of E6AP, an E3 ubiquitinating p53 coerced by the human papillomavirus and regulating pathways implicated in neurodevelopmental disorders such as Angelman syndrome. We found that a γ-AA peptide P6, discovered from the affinity-based screening with the E6AP HECT domain, can significantly stimulate the ubiquitin ligase activity of E6AP to ubiquitinate its substrate proteins UbxD8, HHR23A, and ß-catenin in reconstituted reactions and HEK293T cells. Furthermore, P6 can accelerate the degradation of E6AP substrates in the cell by enhancing the catalytic activities of E6AP. Our work demonstrates the feasibility of using synthetic ligands to stimulate E3 activities in the cell. The E3 stimulators could be developed alongside E3 inhibitors and substrate recruiters such as PROTACs and molecular glues to leverage the full potential of protein ubiquitination pathways for drug development.

A novel cyclic γ-AApeptide-based long-acting pan-coronavirus fusion inhibitor with potential oral bioavailability by targeting two sites in spike protein.

The receptor-binding domain (RBD) in S1 subunit and heptad repeat 1 (HR1) domain in S2 subunit of SARS-CoV-2 spike (S) protein are the targets of neutralizing antibodies (nAbs) and pan-coronavirus (CoV) fusion inhibitory peptides, respectively. However, neither nAb- nor peptide-based drugs can be used orally. In this study, we screened a one-bead-two-compound (OBTC) cyclic γ-AApeptide library against SARS-CoV-2 S protein and identified a hit: S-20 with potent membrane fusion inhibitory activity, but moderate selectivity index (SI). After modification, one derivative, S-20-1, exhibited improved fusion inhibitory activity and SI (>1000). S-20-1 could effectively inhibit infection by pseudotyped and authentic SARS-CoV-2 and pseudotyped variants of concern (VOCs), including B.1.617.2 (Delta) and B.1.1.529 (Omicron), as well as MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63. It could also inhibit infection of a pseudotyped SARS-related coronavirus WIV1 (SARSr-CoV-WIV1) from bats. Intranasal application of S-20-1 to mice before or after challenge with HCoV-OC43 or SARS-CoV-2 provided significant protection from infection. Importantly, S-20-1 was highly resistant to proteolytic degradation, had long half-life, and possessed favorable oral bioavailability. Mechanistic studies suggest that S-20-1 binds with high affinity to RBD in S1 and HR1 domain in S2 of SARS-CoV-2 S protein. Thus, with its pan-CoV fusion and entry inhibitory activity by targeting two sites in S protein, desirable half-life, and promising oral bioavailability, S-20-1 is a potential candidate for further development as a novel therapeutic and prophylactic drug against infection by SARS-CoV-2 and its variants, as well as future emerging and reemerging CoVs.

Small RNA-seq analysis of extracellular vesicles from porcine uterine flushing fluids during peri-implantation.

The extracellular vesicles (EVs) of uterine flushing fluids (UFs) mediate intrauterine communication between conceptus and uterus in pigs. The small RNAs of UFs-EVs are widely recognized as important factors that influence embryonic implantation. However, small RNAs expression profiles of porcine UFs-EVs during peri-implantation are still unknown. In this study, cup-shaped EVs of porcine UFs on days 10 (D10), 13 (D13) and 18 (D18) of pregnancy were isolated and characterized. The expression of small RNAs in these EVs was comprehensively profiled through sequencing. A total of 152 known microRNAs (miRNAs), 43 novel miRNAs, 6248 known Piwi-interacting RNAs (piRNAs) and 110 novel piRNAs were identified. Among these small RNAs, RT-qRCR results indicated that ssc-let-7f-5p, ssc-let-7i-5p and ssc-let-7g were differentially expressed during the three stages. Bioinformatics analysis showed that the miRNAs differentially expressed in the three comparisons (D10 vs D13, D13 vs D18 and D10 vs D18) were involved in important processes and pathways related to immunization, endometrial receptivity and embryo development, which play important roles in embryonic implantation. Our results reveal that EVs from porcine UFs contain various small RNAs with potentially vital effects on implantation. This research also provides resources for studies of miRNAs and piRNAs in the cross-talk between embryo and endometrium.

The folding propensity of α/sulfono-γ-AA peptidic foldamers with both left- and right-handedness.

The discovery and application of new types of helical peptidic foldamers have been an attractive endeavor to enable the development of new materials, catalysts and biological molecules. To maximize their application potential through structure-based design, it is imperative to control their helical handedness based on their molecular scaffold. Herein we first demonstrate the generalizability of the solid-state right-handed helical propensity of the 413-helix of L-α/L-sulfono-γ-AA peptides that as short as 11-mer, using the high-resolution X-ray single crystallography. The atomic level folding conformation of the foldamers was also elucidated by 2D NMR and circular dichroism under various conditions. Subsequently, we show that the helical handedness of this class of foldamer is controlled by the chirality of their chiral side chains, as demonstrated by the left-handed 413-helix comprising 1:1 D-α/D-sulfono-γ-AA peptide. In addition, a heterochiral coiled-coil-like structure was also revealed for the first time, unambiguously supporting the impact of chirality on their helical handedness. Our findings enable the structure-based design of unique folding biopolymers and materials with the exclusive handedness or the racemic form of the foldamers in the future.

Zearalenone Blocks Autophagy Flow and Induces Cell Apoptosis During Embryo Implantation in Gilts.

Zearalenone (ZEA) has been proved to be toxic, particularly to the reproductive system of gilts. The effect of ZEA on gilts during embryo implantation window period is of particular interests. Here, we observed window stage dysontogenesis of gilts treated with ZEA. In endometrial tissues and cells, autophagosomes increased significantly and mitochondria were damaged with increasing ZEA concentration. Addition of autophagy inhibitor confirmed that ZEA blocks the autophagic flow in the fusion of autophagosomes and lysosomes. In conclusion, ZEA exposure during embryo implantation results in endometrium inflammation by activating autophagy while blocking autophagy flow at the same time, leading to the significant accumulation of autophagosomes. The aforementioned effects of ZEA induce the apoptosis of primary endometrial cells through the caspase3 pathway, which would break the uterus environment balance and finally lead to embryo implantation failure and dysontogenesis in gilts.

Sequence analysis of microRNAs during pre-implantation between Meishan and Yorkshire pigs.

Embryonic implantation in sows is a coordinated interaction between the implantation-competent blastocyst and receptive uterus. In addition, microRNAs are small endogenous non-coding RNAs which are involved in post-transcriptional gene regulation of several biological processes including embryonic implantation. However, the mechanisms of miRNAs involved in embryonic implantation of sows remain largely unknown. Here, we analyzed miRNAome of endometrium on day 9, 12 and 15 of pregnancy and on day 12 of non-pregnancy in Meishan and Yorkshire pigs by Illumina sequencing. From 24 libraries, we identified 312 known microRNAs and 211 potential novel miRNAs. Bioinformatics analysis showed that differentially expressed microRNAs on day 12 of pregnancy between the two breeds may play critical roles by involving "p53 signaling pathway" and "Wnt signaling pathway". Furthermore, our results demonstrated that ssc-miR-21, ssc-miR-451, ssc-miR-204, ssc-miR-199a-5p and ssc-miR-199b-5p would play crucial roles for implantation. The data generated in this study were expected to elucidate the influence of microRNAs during pre-implantation in pigs.

Analyses of Long Non-Coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium.

Establishment of implantation in pig is accompanied by a coordinated interaction between the maternal uterine endometrium and conceptus development. We investigated the expression profiles of endometrial tissue on Days 9, 12 and 15 of pregnancy and on Day 12 of non-pregnancy in Yorkshire, and performed a comprehensive analysis of long non-coding RNAs (lncRNAs) in endometrial tissue samples by using RNA sequencing. As a result, 2805 novel lncRNAs, 2,376 (301 lncRNA and 2075 mRNA) differentially expressed genes (DEGs) and 2149 novel transcripts were obtained by pairwise comparison. In agreement with previous reports, lncRNAs shared similar characteristics, such as shorter in length, lower in exon number, lower at expression level and less conserved than protein coding transcripts. Bioinformatics analysis showed that DEGs were involved in protein binding, cellular process, immune system process and enriched in focal adhesion, Jak-STAT, FoxO and MAPK signaling pathway. We also found that lncRNAs TCONS_01729386 and TCONS_01325501 may play a vital role in embryo pre-implantation. Furthermore, the expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy). Our results first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases.