Biology subjects

Tam, J. P.

Publications and source records attributed to Tam, J. P..

6 recordsLinked to original sources

bioRxiv2023

Ginsentide TP1 Protects Hypoxia-Induced Dysfunction and ER Stress-Linked Apoptosis

Hypoxia-induced vascular endothelial dysfunction (VED) is a significant contributor to several severe human conditions, including heart disease, stroke, dementia, and cancer. However, current treatment options for VED are limited due to a lack of understanding of the underlying disease mechanisms and therapeutic leads. We recently discovered a heat-stable microprotein in ginseng, known as ginsentide TP1 that has been shown to reduce vascular dysfunction in cardiovascular disease models. In this study, we use a combination of functional assays and quantitative pulsed SILAC proteomics to determine new proteins synthesized in hypoxia and to show that ginsentide TP1 provides protection for human endothelial cells against hypoxia and ER stress. We found that hypoxia activates various pathways related to endothelium activation and monocyte adhesion, which in turn, impairs nitric oxide (NO) synthase activity, reduces NO bioavailability, and increases the production of reactive oxygen species that contribute to VED. Additionally, hypoxia triggers endoplasmic reticulum stress and initiates apoptotic signaling pathways associated with cardiovascular pathology. Treatment with ginsentide TP1 reduced surface adhesion molecule expression prevented activation of the endothelium and leukocyte adhesion, restored protein hemostasis, and reduced ER stress to protect against hypoxia-induced cell death. Ginsentide TP1 also restored NO signaling and bioavailability, reduced oxidative stress, and protected endothelial cells from endothelium dysfunction. In conclusion, this study shows that the molecular pathogenesis of VED induced by hypoxia can be mitigated by treatment with ginsentide TP1, which could be one of the key bioactive compounds responsible for the "cure-all" effect of ginseng. This research may lead to the development of new therapies for cardiovascular disorders.

pharmacology and toxicology↗

bioRxiv2023

Decoding the Cure-all Effects of Ginseng

Ginseng has been known as a "cure-all" traditional medicine to treat various illnesses and as an adaptogen to relieve stress. However, the known active compounds of ginseng are small-molecule metabolites. Here we report ginsentides, which are disulfide-dense, super-stable and cell-penetrating peptides with 31-33 amino acids, as active compounds and adaptogens that restore homeostasis in response to stress. Using mass spectrometry-based target identification and functional studies, we show that ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via the PI3K/Akt signaling pathway. Ginsentides were also found to alleviate 1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of the aorta, decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40, inhibit P2Y12 receptors, reduce platelet aggregation, and thrombus formation in the lung. Orally administered ginsentides were effective in anti-stress behavior using animal models of tail suspension and forced swimming tests. Together, these results suggest that ginsentides interact with multiple systems to restore homeostasis by reversing stress-induced physiological changes and provide new insights into the panacea medicinal effects of ginseng.

pharmacology and toxicology↗

bioRxiv2022

Consensus design and engineering of an efficient and high-yield Peptide Asparaginyl Ligase

Plant legumains are Asn/Asp-specific endopeptidases (AEPs) that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found AEPs. We first constructed a consensus sequence derived from 1,500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the LAD (ligase-activity determinant) hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a super-efficient and broadly active PAL for peptide and protein cyclization.

biochemistry↗

bioRxiv2022

Substrate-binding Glycine Residues are Major Determinants for Hydrolase and Ligase Activity of Plant Legumains

Peptide asparaginyl ligases (PALs) are Asn/Asp(Asx)-specific ligases that are useful for precision modifications of proteins and live-cell surfaces. However, PALs share high structural similarity to the far more common asparaginyl endopeptidases (AEPs), also known as legumains that hydrolyze peptide bonds after Asx, thus making it challenging to identify PALs in a sea of AEPs. Previously we identified sequences flanking the catalytic site as ligase activity determinants (LADs) for legumains. Here we show that two conserved substrate-binding Gly residues are critical, but negative determinants for ligase activity, based on a combined bioinformatics analysis of 1,500 plant legumains, mutagenesis and functional study of 16 novel legumains, plus identification of seven new PALs. We also show that PALs are rare and AEPs are much more common, accounting for about 1% and 88%, respectively. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies.

plant biology↗

bioRxiv2022

Genomic, transcriptomic, and metabolomic analysis of Traditional Chinese Medicine plant Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites

Natural products from traditional medicinal plants are valuable candidates for clinical cancer therapy. Plants from the Oldenlandia-Hedyotis complex are popular ingredients of Traditional Chinese Medicine (TCM), however a major hurdle in the plant bioprospecting process of TCM plants is that the active metabolites, their biosynthetic pathways, and mode of action are often unknown. We show that Oldenlandia corymbosa extracts are active against breast cancer cell lines. To study the genes involved in the biosynthesis of active compounds in this medicinal plant, we assembled a high-quality genome. We show that the main active compound is ursolic acid and that abiotic stresses cause changes in anti-cancer activity, metabolite composition, and gene expression of plants. To reveal the mode of action of ursolic acid, we show that cancer cells undergo mitotic catastrophe, and we identify three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking.

plant biology↗

bioRxiv2021

Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzymes S1 pocket and why a hydrophobic residue is required at the substrate P2 position. Beside illustrating the role played by P1 and P2 residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities. One sentence summaryWe captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation.

biochemistry↗