Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits -arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

Proteome Profiling Identified Amyloid-β Protein Precursor as a Novel Binding Partner and Modulator of VGLUT1

Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.

The Arg/N-degron pathway targets transcription factors and regulates specific genes

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.

Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network

The Rho guanine nucleotide exchange factor (RGNEF) protein encoded by the ARHGEF28 gene has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Biochemical and pathological studies have shown that RGNEF is a component of the hallmark neuronal cytoplasmic inclusions in ALS-affected neurons. Additionally, a heterozygous mutation in ARHGEF28 has been identified in a number of familial ALS (fALS) cases that may give rise to one of two truncated variants of the protein. Little is known about the normal biological function of RGNEF or how it contributes to ALS pathogenesis. To further explore RGNEF biology we have established and characterized a yeast model and characterized RGNEF expression in several mammalian cell lines. We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein. Additionally, through unbiased screening using the split-ubiquitin system, we have identified RGNEF-interacting proteins, including two ALS-associated proteins. Functional characterization of other RGNEF interactors identified in our screen suggest that RGNEF functions as a microtubule regulator. Our findings indicate that RGNEF misfolding and toxicity may cause impairment of the microtubule network and contribute to ALS pathogenesis.

The ER membrane chaperone Shr3 co-translationally assists biogenesis of related plasma membrane transport proteins

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. In Saccharomyces cerevisiae, Shr3 is an ER membrane-localized chaperone (MLC) that is specifically required for the functional expression of amino acid permeases (AAP), a family of eighteen transporters comprised of 12 MS. Here, comprehensive scanning mutagenesis and deletion analysis of Shr3, combined with a modified split-ubiquitin approach, were used to probe chaperone-substrate (Shr3-AAP) interactions in vivo. A surprisingly low level of sequence specificity in Shr3 underlies Shr3-AAP interactions, which initiate early as the first 2 MS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 MS partition into the membrane. Thus, Shr3 acts transiently in a co-translational manner to prevent MS of AAP translation intermediates from engaging in non-productive interactions, effectively preventing AAP misfolding during biogenesis.

Identification of the Tetraspanin CD9 as an Interaction Partner of Organic Cation Transporters 1 and 2

Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 ( SLC22A1/hOCT1) and hOCT2 ( SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63 and CD9. Moreover, we confirmed interaction of hOCT2 with CD9 by fluorescence-activated cell sorting coupled with Förster resonance energy transfer analysis. Together with other proteins, tetraspanins build “tetraspanins webs” in the plasma membrane, which are able to regulate cellular trafficking and compartmentalization of interacting partners. While CD63 was demonstrated to mediate the localization of the hOCT2 to the endosomal system, we show here that co-expression of hOCT2 and CD9 led to strong cell surface localization of the transporter. These data suggest that tetraspanins regulate the cellular localization and function of OCTs. Co-localization of CD9 and hOCT was confirmed in tissues endogenously expressing proteins, highlighting the potential biological relevance of this interaction.

A pipeline for systematic yeast 2-hybrid matricial screening in liquid culture.

Physical interactions mediated by proteins are a critical element of biological systems, and the analysis of interaction partners can provide valuable hints about unknown functions of a protein. Two major classes of experimental approaches are used for protein interaction mapping: analysis of direct interactions using binary methods such as yeast two-hybrid (Y2H) or split ubiquitin, and analysis of protein complexes through affinity purification followed by mass spectrometry. Thanks to his flexibility to low- and high-throughput approaches and a low operating cost the Y2H assay is widely used for high-throughput interaction mapping experiments. Moreover, it has now been shown that high-throughput methods can produce highly reliable interactome datasets1 2 3 4. Notably, in 2011 a proteome-wide binary protein-protein interaction map of the plant Arabidopsis thaliana 5 (Arabidopsis Interactome Mapping project – AIM) was described using a high-throughput binary interactome mapping pipeline based on the Y2H system and using a collection of ~8,000 open reading frames (8k_space). Here we describe a liquid pipeline for a high-throughput binary protein–protein Y2H screen of a pool of 50 proteins used as baits against a collection of ~12,000 Arabidopsis proteins encoded by sequence-verified ORFs (12k_space)6 7.

The Mitochondrial Ca2+Uniporter Complex (MCUC) ofTrypanosoma bruceiIs a Hetero-oligomer That Contains Novel Subunits Essential for Ca2+Uptake

ABSTRACTThe mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterizedTrypanosoma brucei’s MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralogT. bruceiMCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids.In situtagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria ofT. brucei. Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca2+uptake inT. brucei, respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC ofT. bruceiand defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC.IMPORTANCETrypanosoma bruceicauses human African trypanosomiasis and nagana in animals. The finding of a mitochondrial calcium uniporter (MCU) conserved in this parasite was essential for the discovery of the gene encoding the pore subunit. Mitochondrial Ca2+transport mediated by the MUC complex is critical inTrypanosoma bruceifor shaping the dynamics of cytosolic Ca2+increases, for the bioenergetics of the cells, and for viability and infectivity. We found that one component of the complex (MCUb) does not act as a dominant negative effector of the channel as in vertebrate cells and that the TbMCUC possesses two unique subunits (MCUc and MCUd) present only in trypanosomatids and required for Ca2+transport. The study of the interactions between these four subunits (MCU, MCUb, MCUc, and MCUd) by a variety of techniques that include coimmunoprecipitation, split-ubiquitin membrane-based yeast two-hybrid assays, and site-directed mutagenesis suggests that they interact through their transmembrane helices to form hetero-oligomers.