Cysteine-rich Domain Research Articles

NFATc1-E2F1-LMCD1-Mediated IL-33 Expression by Thrombin Is Required for Injury-Induced Neointima Formation.

Objective- IL (interleukin)-33 has been shown to play a role in endothelial dysfunction, but its role in atherosclerosis is controversial. Therefore, the purpose of this study is to examine its role in vascular wall remodeling following injury. Approach and Results- Thrombin induced IL-33 expression in a time-dependent manner in human aortic smooth muscle cells and inhibition of its activity by its neutralizing antibody suppressed thrombin induced human aortic smooth muscle cell migration but not DNA synthesis. In exploring the mechanisms, we found that Par1 (protease-activated receptor 1), Gαq/11 (Gα protein q/11), PLCβ3 (phospholipase Cβ3), NFATc1 (nuclear factor of activated T cells), E2F1 (E2F transcription factor 1), and LMCD1 (LIM and cysteine-rich domains protein 1) are involved in thrombin-induced IL-33 expression and migration. Furthermore, we identified an NFAT-binding site at -100 nt that mediates thrombin-induced IL-33 promoter activity. Interestingly, we observed that NFATc1, E2F1, and LMCD1 bind to NFAT site in response to thrombin and found that LMCD1, while alone has no significant effect, enhanced either NFATc1 or E2F1-dependent IL-33 promoter activity. In addition, we found that guidewire injury induces IL-33 expression in SMC and its neutralizing antibodies substantially reduce SMC migration and neointimal growth in vivo. Increased expression of IL-33 was also observed in human atherosclerotic lesions as compared to arteries without any lesions. Conclusions- The above findings reveal for the first time that thrombin-induced human aortic smooth muscle cell migration and injury-induced neointimal growth require IL-33 expression. In addition, thrombin-induced IL-33 expression requires LMCD1 enhanced combinatorial activation of NFATc1 and E2F1.

A Frizzled-Like Cysteine-Rich Domain in Glypican-3 Mediates Wnt Binding and Regulates Hepatocellular Carcinoma Tumor Growth in Mice.

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, β-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of β-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

Mapping the chemical and sequence space of the ShKT superfamily

The ShKT superfamily is widely distributed throughout nature and encompasses a wide range of documented functions and processes, from modulation of potassium channels to involvement in morphogenesis pathways. Cysteine-rich secretory proteins (CRISPs) contain a cysteine-rich domain (CRD) at the C-terminus that is similar in structure to the ShK fold. Despite the structural similarity of the CRD and ShK-like domains, we know little of the sequence-function relationships in these families. Here, for the first time, we examine the evolution of the biophysical properties of sequences within the ShKT superfamily in relation to function, with a focus on the ShK-like superfamily. ShKT data were sourced from published sequences in the protein family database, in addition to new ShK-like sequences from the Australian speckled anemone (Oulactis sp.). Our analysis clearly delineates the ShK-like family from the CRDs of CRISP proteins. The four CRISP subclusters separate out into the main phyla of Mammalia, Insecta and Reptilia. The ShK-like family is in turn composed of seven subclusters, the largest of which contains members from across the eukaryotes, with a continuum of intermediate properties. Smaller sub-clusters contain specialised members such as nematode ShK-like sequences. Several of these ShKT sub-clusters contain no functionally characterised sequences. This chemical space analysis should be useful as a guide to select sequences for functional studies and to gain insight into the evolution of these highly divergent sequences with an ancient conserved fold.

Yeast expression of mammalian Onzin and fungal FCR1 suggests ancestral functions of PLAC8 proteins in mitochondrial metabolism and DNA repair

The cysteine-rich PLAC8 domain of unknown function occurs in proteins found in most Eukaryotes. PLAC8-proteins play important yet diverse roles in different organisms, such as control of cell proliferation in animals and plants or heavy metal resistance in plants and fungi. Mammalian Onzin can be either pro-proliferative or pro-apoptotic, depending on the cell type, whereas fungal FCR1 confers cadmium tolerance. Despite their different role in different organisms, we hypothesized common ancestral functions linked to the PLAC8 domain. To address this hypothesis, and to investigate the molecular function of the PLAC8 domain, murine Onzin and fungal FCR1 were expressed in the PLAC8-free yeast Saccharomyces cerevisiae. The two PLAC8-proteins localized in the nucleus and induced almost identical phenotypes and transcriptional changes when exposed to cadmium stress. Like FCR1, Onzin also reduced DNA damage and increased cadmium tolerance by a DUN1-dependent pathway. Both proteins activated transcription of ancient mitochondrial pathways such as leucine and Fe-S cluster biosynthesis, known to regulate cell proliferation and DNA repair in yeast. These results strongly suggest a common ancestral function of PLAC8 proteins and open new perspectives to understand the role of the PLAC8 domain in the cellular biology of Eukaryotes.

A trypsin inhibitor-like protein secreted by Cotesia vestalis teratocytes inhibits hemolymph prophenoloxidase activation of Plutella xylostella

To establish successful infections, endoparasitoid wasps must develop strategies to evade immune responses of the host. Here, we identified and characterized a teratocytes-expressed gene encoding a trypsin inhibitor-like protein containing a cysteine-rich domain from Cotesia vestalis, CvT-TIL. CvT-TIL had a high expression level during the later developmental stage of teratocytes and was secreted into host hemolymph. Further experiments showed CvT-TIL strongly suppressed the prophenoloxidase activation of host hemolymph in a dose-dependent manner by interacting with PxPAP3 of PO cascade. Our results not only provide evidence for an inhibition between CvT-TIL gene and the host’s melanization activity, but also expand our knowledge about the mechanisms by which parasitoids regulate humoral immunity of the host.

Functional Characterization of a Type 4 Metallothionein Gene (CsMT4) in Cucumber

The excess amounts of heavy metals are toxic to various physiological processes in plants. Plant metallothioneins (MTs) are low molecular weight, cysteine-rich metal-binding proteins that play important roles in the detoxification of heavy metal ions. In this study, we characterized CsMT4, a plant type 4 MT gene identified in a complementary DNA (cDNA) library prepared from young cucumber (Cucumis sativus) fruit. CsMT4 encodes a 90 amino acid protein with a predicted molecular mass of 9.028 kD. CsMT4 contains 17 cysteine residues in three highly conserved cysteine-rich domains. In contrast to the structures of other MTs, the highly conserved amino acid pattern CxCxxxCxCxxCxC is present in the middle of CsMT4. Furthermore, CsMT4 was markedly induced in various tissues by various concentrations of Cd2+ and Zn2+ (> 1.0 mmol•L−1). Similar to AtMT4b function, heterologous expression of CsMT4 in E. coli could also improved its tolerance to Cd2+ and led to increased uptake of Cd2+, and the rate of Cd2+ uptake was the highest in cells expressing a phytochelatin-like peptide. Our findings demonstrate that CsMT4 might be obviously induced by the metal stress in cucumber, and improves tolerance to Cd ions but not Zn ions when heterologously expressed in E. coli, and suggest that the composition and arrangement of N-terminal Cys-residues in MT4 are associated with their binding capacity and preference for different metal ions.

SAT-036 Extracellular Domain Calcium-Sensing Receptor (CaSR) Mutations Leading to Hypercalcemic and Hypocalcemic Disorders Cluster at the Homodimeric Interface

The calcium-sensing receptor (CaSR) is a homodimeric G-protein coupled receptor that plays a pivotal role in extracellular Ca2+ (Ca2+e) homeostasis, and loss- and gain-of-function mutations of the CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively. Crystal structure analysis has shown that the CaSR has a large extracellular domain (ECD) comprising lobes 1 and 2, and a cysteine-rich domain (CRD), which connects the ECD to the transmembrane domain. The CaSR ECD binds Ca2+e at multiple sites, which induces conformational changes at the extracellular dimer interface that lead to intracellular signaling via the inositol trisphosphate (IP3) and mitogen-activated protein kinase (MAPK) pathways. To further elucidate the structure-function relationships of the ECD, we examined the location of all CaSR ECD mutations reported to-date in FHH1 and ADH1 probands using available CaSR crystal structures. These studies identified that 127 FHH1 and 66 ADH1 mutations affected ECD residues, with >50% of these mutations being located at the dimer interface. Mutations predicted to disrupt key CaSR dimer-dimer interactions included: a lobe 1 Tyr161Cys mutation, which impaired an interprotomer interaction with the lobe 1 Pro55 residue; a Ser171Asn mutation predicted to disrupt a lobe 2 interprotomer salt bridge, which forms upon agonist binding; and a Gly553Arg mutation, which altered interprotomer hydrophobic interactions within the CRD. We investigated the effect of the Tyr161Cys, Ser171Asn and Gly553Arg mutations on receptor dimerization using western blotting and showed that these mutant proteins all form dimers similar to the wild-type (WT) CaSR. Furthermore, we evaluated the effect of these mutations on CaSR function in HEK293 cells following stimulation with Ca2+e, by measuring accumulation of inositol monophosphate (IP1), which is an IP3 metabolite, and also by measuring serum response element (SRE)-containing luciferase reporter fold-change responses, which is an indicator of MAPK activation. The Tyr161Cys, Ser171Asn and Gly553Arg dimer interface mutations were shown to significantly impair CaSR-mediated signaling. Indeed, cells expressing the Tyr161Cys, Ser171Asn or Gly553Arg mutant CaSRs all showed a >30% reduction in IP1 accumulation and >50% reduction in SRE-reporter responses, respectively, compared to WT-expressing cells (p<0.05, results are from 3 independent experiments). Thus, these studies demonstrate that the majority of FHH1- and ADH1-causing CaSR ECD mutations are located at the dimer interface, but likely do not disrupt dimer formation. Instead, these mutations may potentially influence conformational changes that occur at the dimer interface upon CaSR activation.

Insights into Molecular Interactions of human Wnt5b and Frizzled proteins for their role in teratogenicity.

Wnt-Fzd signalling plays vital role in different physiological pathways including embryonic development and supposed to be probable target of many teratogens. The present study was done to investigate the role of human Wnt5b interaction with different isoforms of human Fzds and also the molecular interactions of their complexes with selected known teratogens [Carbamazepine (CBZ), Retinoic acid (RA), Valproic acid (VPA), Aminopterin (AMP) and Phenytoin (PHY)] using Niclosamide (NLM) as standard. The models of hWnt5b and hFzd isoforms, whose solved crystal structures were unavailable, were generated using homology modeling and hWnt5b was subjected to protein-protein docking studies against different isoforms of hFzd. The macromolecular docking studies of hWnt5b-hFzds complexes revealed that hWnt5b had highest binding affinity with hFzd8 and lowest with hFzd1, respectively. The Cysteine rich domain (CRD) of hFzds docked against hWnt5b into a palm shaped opening or near the largest binding pocket as in hWnt5b-hFzd6. The possible role of Wnt-Fzd interactions in developmental toxicity due to selected teratogens were also investigated using molecular docking studies which showed that Retinoic Acid possessed the maximum binding affinity with binding energy of for hWnt5b-hFzd8 complex while VPA was observed to have lowest binding affinity towards all the studied hWnt5b-hFzd complexes.

Identification and function of penaeidin 3 and penaeidin 5 in Fenneropenaeus merguiensis

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.

Lysyl Oxidase Like 2 Processing by Factor Xa Modulates its Activity and Localization

IntroductionLysyl oxidase like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family of amine oxidases that catalyzes the oxidative deamination of lysine residues to crosslink extracellular matrix (ECM) proteins, thereby forming a stable and highly organized matrix. LOXL2 is shown to be involved in a diverse set of diseases including hepatic fibrosis and cancer metastasis, due to its critical role in tubulogenesis, matrix assembly, and angiogenesis. Despite the significant advances made in linking LOXL2 to fibrosis initiation and disease progression, the mechanisms of LOXL2 regulation are still largely unknown.Prior studies have shown that LOXL2 is processed extracellularly and LOXL2 processing putatively regulates its catalytic function. However, the exact identity of the protease that processes LOXL2 remains unknown. Specifically, LOXL2 is composed of a C‐terminal prototypic LOX domain that has the enzymatic function to catalyze ECM protein crosslinking, and four N‐terminal scavenger receptor cysteine rich (SRCR) domains, which are typically involved in protein‐protein recognition and signaling. Extracellular processing releases an N‐terminus fragment of 317 amino acids containing SRCR domains 1–2 and a C‐terminus fragment of SRCR domains 3–4 along with the LOX domain.Methods and ResultsIn silico digest of LOXL2 using the ExPASy peptide cutter showed Factor Xa as the potential protease that cleaves LOXL2 at position 317. Therefore, we first tested processing of exogenously expressed LOXL2 in cell culture media in vitro. Factor Xa dose dependently increased LOXL2 processing, and this was inhibited using aprotinin (non‐specific inhibitor) and rivaroxaban (specific inhibitor). We next determined the effect of LOXL2 processing on its sub‐cellular localization. Comparing the full‐length and processed LOXL2 content in the conditioned cell‐culture media and ECM showed that full length LOXL2 is predominantly found in the ECM and the processed form is found predominantly in the media. Next, we investigated whether LOXL2 processing regulates its catalytic activity. To this end, we measured the in‐situ assembly of FITC‐labeled collagen I or elastin, the main substrates of LOXL2, by processed and full length LOXL2. The incorporation of collagen I/elastin and the deposition of LOXL2 were visualized by confocal epifluorescence microscopy. Our findings indicate that LOXL2 processing markedly reduces collagen I/elastin incorporation when compared to that by full‐length LOXL2.ConclusionsFactor Xa is the protease responsible for LOXL2 processing in vitro. LOXL2 processing inhibits its ability to utilize collagen I and elastin as substrates. Future studies will continue to 1) investigate whether LOXL2 processing regulates its substrate specificity by shifting LOXL2 catalysis to Collagen IV, 2) determine the functions of the cleaved SRCR1–2 propeptide, 3) identify the specific mechanism by which processed and full length LOXL2 are trafficked to the cell‐derived ECM, and 4) evaluate the in vivo significance of this processing in the vascular compartment.Support or Funding InformationFunded by JHMI ACCM StAAR Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mice conditionally expressing RET(C618F) mutation display C cell hyperplasia and hyperganglionosis of the enteric nervous system.

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.

A four-gene signature for prognosis in breast cancer patients with hypermethylated IL15RA.

Previous studies have revealed that upregulation of interleukin 15 receptor α (IL15RA) contributes to improved prognosis of breast cancer. The present study aimed to elucidate the molecular mechanisms underlying the antitumor effect induced by IL15RA upregulation, and to identify a gene signature capable of predicting the survival of patients with breast cancer. Using paired gene expression and methylation data of breast cancer samples from The Cancer Genome Atlas data portal, differentially expressed genes (DEGs) were identified in hypermethylated and hypomethylated IL15RA breast cancer samples. Furthermore, a gene signature-based risk-scoring model was developed according to the Cox regression coefficients of survival-associated DEGS. The gene signature was applied to classify patients with breast cancer and hypermethylated IL15RA into two risk groups via Kaplan-Meier survival analysis of overall survival (OS) time. Functional enrichment analysis was conducted to decipher the biological roles of the DEGs between the two risk groups. A total of 326 DEGs were present in the hypomethylation and hypermethylation samples compared with in the normal samples. A four-gene signature [SH3 and cysteine rich domain 2 (STAC2), proline rich 11 (PRR11), homeobox C11 (HOXC11) and nucleolar and spindle associated protein 1 (NUSAP1)] was identified as able to successfully separate patients with breast cancer and hypermethylated IL15RA into two risk groups with significantly different OS time. The signature revealed similar predictive performance in an independent set. Significant enrichment of the ‘receptor interaction’ and ‘cell adhesion molecules (CAM)’ pathways, which involved the DEGs, occurred between the two risk groups. These findings suggested that IL15RA may participate in the regulation of STAC2, PRR11, HOXC11, NUSAP1, and ‘ECM-receptor interaction’ and ‘cell adhesion molecules’ pathways, and therefore in the suppression of breast cancer development and progression. The four-gene signature may have potential prognostic value for breast cancer.

Structural characterization of the third scavenger receptor cysteine-rich domain of murine neurotrypsin.

Neurotrypsin (NT) is a multi-domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C-terminal serine-protease (SP) domain. However the purpose of NT's remaining 3-4 scavenger receptor cysteine-rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT-SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca2+ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.

Molecular characterization, recombinant expression and bioactivity profile of an antimicrobial peptide, Ss-arasin from the Indian mud crab, Scylla serrata

Antimicrobial peptides (AMP) are potential alternatives to conventional antibiotics with the prospect to treat infections caused by multidrug resistant bacteria. This is the report of the first arasin sequence from the mud crab, Scylla serrata, designated as Ss-arasin. The complete cDNA sequences of the open reading frame (ORF) is comprised of 198 bp encoding 65 amino acid with a predicted molecular weight of 7 kDa and a predicted isoelectric point of 10.68. The sequence of the N-terminal 24 amino acid residues is indicative of a signal sequence directing the newly synthesize protein toward the secretory pathway. The 41-residue mature peptide is composed of two domains, an N-terminal Gly/Arg-rich domain and a C-terminal cysteine-rich domain. Challenging the mud crab with lipopolysaccharide (LPS) increased expression of Ss-arasin mRNA in haemocytes, reaching the highest level at 6 h, before dropping to basal levels at 24 h. Recombinant rSs-arasin showed antimicrobial activity against three bacterial species Staphylococcus aureus (40 mM), Pseudomonas aeruginosa (40 mM) and Escherichia coli (40 mM) implying significant anti-bacterial action. In addition, recombinant rSs-arasin inhibited human cervical carcinoma (HeLa) and colon carcinoma (HT-29) cell growth. These initial findings are encouraging to further study the structure-activity relationships to optimize these biological functions for future drug development.

Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2)

Receptor tyrosine kinase like orphan receptor 2 (ROR2) is a co-receptor for some Wnt proteins including Wnt5a that activate the noncanonical Wnt/planar cell polarity (PCP) signaling pathway. Upregulation of ROR2 is associated with several cancer forms. The extracellular region of ROR2, which contains an immunoglobulin (Ig)-like domain, a Frizzled like cysteine-rich domain (CRD) and a Kringle domain, is a potential anticancer drug target. The structural and biochemical properties of the ROR2 extracellular region remain largely unexplored. Here we describe the mapping and purification, using a baculovirus - insect cell system, of a near-full-length ROR2 extracellular fragment (residues 53–402), which is well-behaved and suitable for future structural and biochemical analysis. We show that the extracellular region of ROR2 per se is monomeric in solution. Different monoclonal antibodies raised against the purified ROR2 protein can specifically recognize the protein and can either inhibit or activate the PCP activity in a cell-based assay, and are thus potentially useful for future mechanistic and therapeutic/diagnostic studies. The biological relevance of these antibodies further demonstrates that the purified recombinant ROR2 protein is properly folded and biochemically active.

Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits

Class C G protein-coupled receptors (GPCRs) are obligatory dimers that are particularly important for neuronal responses to endogenous and environmental stimuli. Ligand recognition through large extracellular domains leads to the reorganization of transmembrane regions to activate G protein signaling. Although structures of individual domains are known, the complete architecture of a class C GPCR and the mechanism of interdomain coupling during receptor activation are unclear. By screening a mutagenesis library of the human class C sweet taste receptor subunit T1R2, we enhanced surface expression and identified a dibasic intracellular retention motif that modulates surface expression and co-trafficking with its heterodimeric partner T1R3. Using a highly expressed T1R2 variant, dimerization sites along the entire subunit within all the structural domains were identified by a comprehensive mutational scan for co-trafficking with T1R3 in human cells. The data further reveal that the C terminus of the extracellular cysteine-rich domain needs to be properly folded for T1R3 dimerization and co-trafficking, but not for surface expression of T1R2 alone. These results guided the modeling of the T1R2-T1R3 dimer in living cells, which predicts a twisted arrangement of domains around the central axis, and a continuous folded structure between transmembrane domain loops and the cysteine-rich domains. These insights have implications for how conformational changes between domains are coupled within class C GPCRs.

Crystal structure of the m4-1BB/4-1BBL complex reveals an unusual dimeric ligand that undergoes structural changes upon 4-1BB receptor binding

The interaction between the receptor 4-1BB and its ligand 4-1BBL provides co-stimulatory signals for T-cell activation and proliferation. However, differences in the mouse and human molecules might result in differential engagement of this pathway. Here, we report the crystal structure of mouse 4-1BBL and of the mouse 4-1BB/4-1BBL complex, which together provided insights into the molecular mechanism by which m4-1BBL and its cognate receptor recognize each other. Unlike all human or mouse tumor necrosis factor ligands that form noncovalent and mostly trimeric assemblies, the m4-1BBL structure formed a disulfide-linked dimeric assembly. The structure disclosed that certain differences in the amino acid composition along the intramolecular interface, together with two specific residues (Cys-246 and Ser-256) present exclusively in m4-1BBL, are responsible for this unique dimerization. Unexpectedly, upon m4-1BB binding, m4-1BBL undergoes structural changes within each protomer; moreover, the individual m4-1BBL protomers rotate relative to each other, yielding a dimerization interface with more inter-subunit interactions. We also observed that in the m4-1BB/4-1BBL complex, each receptor monomer binds exclusively to a single ligand subunit with contributions of cysteine-rich domain 1 (CRD1), CRD2, and CRD3. Furthermore, structure-guided mutagenesis of the binding interface revealed that novel binding interactions with the GH loop, rather than the DE loop, are energetically critical and define the m4-1BB receptor selectivity for m4-1BBL. A comparison with the human 4-1BB/4-1BBL complex highlighted several differences between the ligand- and receptor-binding interfaces, providing an explanation for the absence of inter-species cross-reactivity between human and mouse 4-1BB and 4-1BBL molecules.

Raf-1 Cysteine-Rich Domain (CRD) Promotes Active Orientation and Dimerization of KRas4B at the Membrane

Raf-1 Cysteine-Rich Domain (CRD) Promotes Active Orientation and Dimerization of KRas4B at the Membrane

Evaluation of differential qPE9-1/DEP1 protein domains in rice grain length and weight variation

BackgroundqPE9–1/DEP1, encoding a G protein γ subunit, has multiple effects on plant architecture, grain size, and yield in rice. The qPE9–1 protein contains an N-terminal G gamma-like (GGL) domain, a putative transmembrane domain, and a C-terminal cysteine-rich domain. However, the roles of each domain remain unclear.ResultsIn the present study, we focused on the genetic effects of different domains of qPE9–1 in the regulation of grain length and weight. We generated a series of transgenic plants expressing different truncated qPE9–1 proteins through constitutive expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 strategies. Phenotypic analysis indicated that the complete or long-tailed qPE9–1 contributed to the elongation of grains, while the GGL domain alone and short-tailed qPE9–1 led to short grains. The long C-terminus of qPE9–1 including two or three C-terminal von Willebrand factor type C domains effectively repressed the negative effects of the GGL domain on grain length and weight. qPE9–1-overexpressing lines in a Wuxianggeng 9 (carrying a qpe9–1 allele) background showed increased grain yield per plant, but lodging occurred in some years.ConclusionsManipulation of the C-terminal length of qPE9–1 through genetic engineering can be used to generate varieties with various grain lengths and weights according to different requirements in rice breeding. The genetic effects of qPE9–1/qpe9–1 are multidimensional, and breeders should take into account other factors including genetic backgrounds and planting conditions in the use of qPE9–1/qpe9–1.

The main WAP isoform usually found in camel milk arises from the usage of an improbable intron cryptic splice site in the precursor to mRNA in which a GC-AG intron occurs

BackgroundWhey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The organization of the eutherian WAP genes is highly conserved through evolution. It has been proposed that WAP could play an important role in regulating the proliferation of mammary epithelial cells. A bacteriostatic activity was also reported. Conversely to the other mammalian species expressing WAP in their milk, camel WAP contains 4 additional amino acid residues at the beginning of the second 4-DSC domain, introducing a phosphorylation site. The aim of this study was to elucidate the origin of this specificity, which possibly impacts its physiological functions.ResultsUsing LC-ESI-MS, we identified in Camelus bactrianus from Kazakhstan a phosphorylated whey protein, exhibiting a molecular mass (12,596 Da), 32 Da higher than the original WAP (12,564 Da) and co-eluting with WAP. cDNA sequencing revealed a transition G/A, which modifies an amino acid residue of the mature protein (V12 M), accounting for the mass difference observed between WAP genetic variants. We also report the existence of two splicing variants of camel WAP precursors to mRNA, arising from an alternative usage of the canonical splice site recognized as such in the other mammalian species. However, the major camel WAP isoform results from the usage of an unlikely intron cryptic splice site, extending camel exon 3 upstream by 12-nucleotides encoding the 4 additional amino acid residues (VSSP) in which a potentially phosphorylable Serine residue occurs. Combining protein and cDNA sequences with genome data available (NCBI database), we report another feature of the camel WAP gene which displays a very rare GC-AG type intron. This result was confirmed by sequencing a genomic DNA fragment encompassing exon 3 to exon 4, suggesting for the GC donor site a compensatory effect in terms of consensus at the acceptor exon position.ConclusionsCombining proteomic and molecular biology approaches we report: the characterization of a new genetic variant of camel WAP, the usage of an unlikely intron cryptic splice site, and the occurrence of an extremely rare GC-AG type of intron.