Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

The Hedgehog (Hh) signaling pathway is critical for embryonic development and tissue renewal. The G protein-coupled receptor (GPCR)-like protein Smoothened (SMO) is the central signal transducer in the Hh pathway. Cholesterol binds and then covalently links to the D95 residue of cysteine-rich domain (CRD) of human SMO. The cholesterylation of CRD is critical for SMO activation. SMO cholesterylation is a Ca 2+-boosted autoreaction that requires the formation of an ester bond between the side chains of D95 and Y130 as an intermediate. It is unknown whether other residues of SMO are involved in the esterification between D95 and cholesterol. In this study, we find that the SMO-CRD(27-192) can undergo cholesterylation. In addition to D95 and Y130, the residues critical for cholesterol modification include Y85, T88, T90, W109, W119, K133, E160 and F166. T88, W109, W119 and F166 also seem to be involved in protein folding. Notably, we find that Y85 and K133 form a cation-π interaction whose disruption abolishes cholesterylation and ciliary localization of SMO. This study highlights the mechanism and function of cholesterol modification of SMO.

In the yeast Saccharomyces cerevisiae and other ascomycetes, the maintenance of cell wall integrity is governed by a family of plasma-membrane spanning sensors that include the Wsc-type proteins. These cell wall proteins apparently sense stress-induced mechanical forces at the cell surface and target the cell wall integrity (CWI) signaling pathway, but the structural base for their sensor function is yet unknown. Here, we solved a high-resolution crystal structure of the extracellular cysteine-rich domain (CRD) of yeast Wsc1, which shows the characteristic PAN/Apple domain fold with two of the four Wsc1 disulfide bridges being conserved in other PAN domain cores. Given the general function of PAN domains in mediating protein-protein and protein-carbohydrate interactions, this finding underpins the importance of Wsc domains in conferring sensing and localization functions. Our Wsc1 CRD structure reveals an unusually high number of surface-exposed aromatic residues that are conserved in other fungal CRDs, and can be arranged into three solvent-exposed clusters. Mutational analysis demonstrates that two of the aromatic clusters are required for conferring S. cerevisiae Wsc1-dependent resistance to the glucan synthase inhibitor caspofungin, and the chitin-binding agents Congo red and Calcofluor white. These findings suggest an essential role of surface-exposed aromatic clusters in fungal Wsc-type sensors that might include an involvement in stress-induced sensor-clustering required to elicit appropriate cellular responses via the downstream CWI pathway.

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells, reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer.

The receptor tyrosine kinase RET is essential in a variety of cellular processes. RET gain-of-function is strongly associated with several cancers, notably multiple endocrine neoplasia type 2A (MEN 2A), while RET loss-of-function causes Hirschsprung\'s disease and Parkinson\'s disease. To investigate the activation mechanism of RET as well as to enable drug development, over-expressed recombinant protein is needed for in vitro functional and structural studies. By comparing insect and mammalian cells expression of the RET extracellular domain (RETECD), we showed that the expression yields of RETECD using both systems were comparable, but mammalian cells produced monomeric functional RETECD, whereas RETECD expressed in insect cells was non-functional and multimeric. This was most likely due to incorrect disulfide formation. By fusing an Fc tag to the C-terminus of RETECD, we were able to produce, in HEK293T cells, dimeric oncogenic RETECD (C634R) for the first time. The protein remained dimeric even after cleavage of the tag via the cysteine disulfide, as in full-length RET in the context of MEN 2A and related pathologies. Our work thus provides valuable tools for functional and structural studies of the RET signaling system and its oncogenic activation mechanisms.

Osteoarthritis (OA) is a chronic inflammatory joint disease without effective drugs. Frizzled 7 (FzD7) binds its ligand Wnt3a through an extracellular cysteine-rich domain (CRD) to transduce the canonical Wnt/β-catenin signaling pathway, which has been strongly implicated in OA pathogenesis. Effects of recombinant protein of FzD7 CRD on Wnt/β-catenin signaling and chondral destruction was evaluated in this study. Firstly, increased protein levels of FzD7, Wnt3a and β-catenin were detected in human OA cartilage implying that the canonical Wnt/β-catenin signaling mediated by Wnt3a and FzD7 executes an essential role in OA. Then we showed that FzD7 CRD antagonized the Wnt3a/β-catenin signaling pathway in a dose-dependent manner by binding Wnt3a. In addition, FzD7 CRD increased the expression of glycosaminoglycans (GAGs), Collagen II, aggrecan and reduced the expression of matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in Wnt3a-stimulated human chondrocytes. Furthermore, a single intra-articular injection of the FzD7 CRD was efficacious in destabilization of the medial meniscus (DMM) mouse OA model, significantly improving Osteoarthritis Research Society International (OARSI) histology scores compared to mice treated with PBS. The results indicate that the FzD7 CRD exhibits chondroprotective effects by binding Wnt3a to suppress the Wnt3a/β-catenin signaling. Targeting the FzD7 CRD may be a novel therapy for the treatment of OA.

Several proteins other than the frizzled receptors (Fzd) and the secreted Frizzled-related proteins (sFRP) contain Fzd-type cysteine-rich domains (CRD). We have termed these domains \"putative Fzd-type CRDs\", as the relevance of Wnt signalling in the majority of these is unknown; the RORs, an exception to this, are well known for mediating non-canonical Wnt signalling. In this study, we have predicted the likely binding affinity of all Wnts for all putative Fzd-type CRDs. We applied both our previously determined Wnt‒Fzd CRD binding affinity prediction model, as well as a newly devised model wherein the lipid term was forced to contribute favourably to the predicted binding energy. The results obtained from our new model indicate that certain putative Fzd CRDs are much more likely to bind Wnts, in some cases exhibiting selectivity for specific Wnts. The results of this study inform the investigation of Wnt signalling modulation beyond Fzds and sFRPs.