Four cysteine residues in the Plasminogen-Apple-Nematode domain are critical for HGF/c-MET signaling

PAN domains have significant functional versatility fulfilling diverse biological roles by mediating protein-protein and protein-carbohydrate interactions. These domains contain a hair-pin loop like structure, similar to that found in knottins but with a different pattern of disulfide bonds. The PAN domain is shared by the plasminogen/hepatocyte growth factor (HGF) protein family, the prekallikrein/coagulation factor XI protein family, and nematode proteins. The domain possesses the characteristic 4-6-cysteine residues in its core that are strictly conserved. These cysteine residues have been proposed to engage in two or three disulfide bridges to form a hairpin loop structure. The PAN domain has been suggested to mediate protein-protein or carbohydrate-protein interactions and facilitate receptor dimerization. HGF is secreted by mesenchymal cells as a single-chain, biologically inert precursor and is converted into its bioactive form when extracellular proteases cleave the bond between Arg494 and Val4957. The mature form of HGF consists of an α- and β-chain, which are held together by a disulfide bond. The α-subunit of HGF includes N-terminal hairpin loop structure and four kringle domains (K1-K4) whereas the β-subunit consists of serine protease homology (SPH) domain.

To provide evidence for its role in cellular mesenchymal-epidermal transition (c-MET) interaction and signal transduction, the authors characterized the functional role of the PAN domain in the heparin binding glycoprotein HGF, which functions as a ligand for the high-affinity receptor, c-MET. HGF/c-MET signaling has been shown to mediate cellular processes including angiogenesis, anti-apoptosis, mitogenesis, morphogenesis, mitogenesis and neurite extension. Dysregulation of the HGF/c-MET signaling cascade can lead to tumorigenesis by transforming normal cells into tumor cells and c-MET hyperactivation has been reported in cancers including lung cancer, colorectal cancer, glioblastoma, and acute myeloid lymphoma among others. A crucial step in HGF/c-MET cascade activation is that the binding of HGF to c-MET brings the dimerization of c-MET that enables its intracellular kinase domains to undergo autophosphorylation. The phosphorylated kinase domain recruits downstream cytosolic effector proteins, leading to the activation of downstream signaling pathways. The HGF/c-MET pathway has emerged as a prime target for cancer pathways and tumorigenesis. However, despite the clinical therapeutic significance of this pathway, the mechanism by which HGF activates c-MET is not well understood. HGF is a bivalent ligand with high-affinity binding pockets in the α chain and relatively low-affinity binding pockets in the β chain. Studies demonstrated that the N- domain and the first kringle domain are enough for c-MET binding; however, for activation of c-MET, the β chain is essential. Although the affinity of the α chain of HGF and the SEMA domain of MET is structurally well-characterized and functionally validated, it is not clear what regions of the α chain bind with c-MET.

Researchers at the Department of Energy’s Oak Ridge National Laboratory led by Dr. Wellington Muchero have definitively linked the function of a specific domain of proteins important in plant-microbe biology to a cancer trigger in humans, knowledge that had eluded scientists for decades. The team’s findings, published in Communications Biology, open up a new avenue for the development of selective drug therapies to fight a variety of cancers such as breast and stomach cancer.

ORNL scientists set out to prove experimentally what they first deduced with computational studies: that the plasminogen-apple-nematode, or PAN, domain is linked to the cell proliferation that drives tumor growth in humans and defense signaling during plant-microbe interactions in bioenergy crops. The association was first made as researchers explored the genomes of crops like poplar and willow. The authors pinpointed four core amino acids called cysteine residues in the HGF protein critical to the PAN domain’s function and studied their behavior in human cancer cell lines. They found that mutating any one of those amino acids turned off the signaling pathway known as HGF-c-MET that is abnormally heightened in cancer cells, causing them to rapidly multiply and spread. Since cysteine residues are known to have many functions, the scientists also randomly tested other cysteines throughout the protein and found that none of them had the same impact on shutting down HGF-c-MET signaling. Mutating the four key cysteines had no effect on the overall structure of the protein, and merely inhibited the cancer signaling pathway, the team noted in the study.

It’s very difficult to engineer molecules to interfere with an entire protein therefore, knowing the specific amino acids to target within that protein is a big advancement. Therefore, is no need to search the entire protein; just these four specific residues are sufficient.