Formation Of Heteromeric Complexes Research Articles

Molecular characterization and function of the lipid raft protein Lvflotillin-1A from Litopenaeus vannamei

White spot syndrome virus (WSSV) can cause a contagious, high virulent and pandemic disease for crustaceans, especially shrimps. However, the molecular mechanism of WSSV pathogenesis remains unclear. Flotillins are lipid raft-associated proteins, which mainly include flotillin-1 and flotillin-2. They are involved in the formation of large heteromeric protein complexes engaged in diverse signalling pathways at the membrane-cytosol interface. They defined a clathrin-independent endocytic pathway in mammalian cells. Our previous studies suggested that shrimp flotillin-2 might mediate endocytosis involved in WSSV infection. To further explore the function of shrimp flotillin, a flotillin-1 homologous, Lvflotillin-1A was identified and characterized in Litopenaeus vanamei. The transcription of Lvflotillin-1A showed a significant decline at 12h post-infection, followed by complete recovery and a slight up-regulation after the WSSV challenge. Gene silencing revealed that inhibition of Lvflotillin-1A raised the virus infection, suggesting Lvflotillin-1A might play an important role in shrimp immunity. Furthermore, co-immunoprecipitation and immunofluorescence illustrated that Lvflotillin-1A and Lvflotillin-2 could form hetero-oligomers, and co-expression promoted the accumulation of intracellular vesicles. The study revealed that WSSV might up-regulate Lvflotillin-2 expression and alter the subcellular location of Lvflotillin-1 protein to facilitate virus infection. These results will provide information for understanding the interaction between WSSV and shrimp.

Heteromeric interactions of ripening-related ethylene receptors in tomato fruit.

Ripening of climacteric fruits is initiated when the gaseous plant hormone ethylene is perceived by the cell. Ethylene binding to membrane-associated ethylene receptors (ETRs) triggers a series of biochemical events through multiple components, resulting in the induction of numerous ripening-related genes. In tomato (Solanum lycopersicum L.), there are seven members of the ETR family, which each contribute to the regulation of fruit ripening. However, the relative contribution of each individual receptor to ethylene signaling remains unknown. Here, we demonstrated the formation of heteromeric receptor complexes across the two ETR subfamilies in tomato fruit. Immunoprecipitation of subfamily II SlETR4 resulted in co-purification of subfamily I (SlETR1, SlETR2, and SlETR3), but not subfamily II members (SlETR5, SlETR6, and SlETR7). Such biased interactions were verified in yeast two-hybrid assays, and in transgenic Arabidopsis plants, in which heterologous SlETR4 interacts with subfamily I ETRs. Our analysis also revealed that the receptor complexes engage the Raf-like protein kinases SlCTR1 and SlCTR3, which are potential regulators of signaling. Here, we suggest that tomato receptor members form heteromeric complexes to fine-tune signal output to the downstream pathway, which is similar to that of the Arabidopsis system but appears to be partially diverged.

α1B/D-adrenoceptors regulate chemokine receptor–mediated leukocyte migration via formation of heteromeric receptor complexes

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

CYP1A1 and CYP1A2 Form Distinct Protein Complexes with NADPH‐cytochrome P450 Reductase, Cytochrome b5, and Heme Oxygenase‐1

The cytosolic face of the endoplasmic reticulum (ER) membrane contains a variety of enzymes involved in oxidative metabolism. Among these, NADPH‐cytochrome P450 reductase (POR) and cytochrome b5 (CYB5) provide reducing electrons necessary for cytochrome P450 (P450) and heme oxygenase‐1 (HO‐1) activity. HO‐1 catalyzes the first and rate‐limiting step in free heme catabolism while the P450s are responsible for the metabolism of endogenous and exogenous compounds, including a majority of prescribed drugs. P450s have been shown to form heteromeric and homomeric complexes with other P450s and these complexes have been associated with alterations in P450 activity. Additionally, the P450 catalytic cycle requires 2 electrons—while POR is able to provide both, CYB5 can provide the second electron in some cases. However, binding of POR and CYB5 to the same P450 has been reported to be mutually exclusive, raising the question of how CYB5 can stimulate P450 activity if POR must dissociate prior to CYB5 binding.In order to shed light on this question, we set out to collect more information on the protein‐protein interaction network that the ER‐membrane proteins participate in. Using bioluminescence resonance energy transfer (BRET), we are able to detect interactions between a pair of proteins and, further, to measure the stability of that pair in the presence of a third protein. To detect protein‐protein interactions, HEK293T cells were co‐transfected with vectors encoding two potentially interacting proteins tagged with either Renilla luciferase (Rluc) or GFP2.While human CYP1A2 showed evidence of a specific complex, with analysis of BRET data showing the presence of homodimers, human CYP1A1 did not form a specific homomeric complex. CYP1A1 and CYP1A2 also differed in how they interacted with other ER membrane proteins. Both proteins showed evidence of heteromeric complex formation with HO‐1, but CYP1A1 only did so in the presence of excess POR. POR and CYB5 do not appear to interact with each other and in the presence of CYP1A2, HO‐1, and several other P450s, a complex between POR and CYB5 was not detected. However, a complex including the two proteins was detected in the presence of CYP1A1 which indicated the presence of a ternary CYB5‐POR‐CYP1A1 complex. The presence of a ternary complex could explain the stimulation of P450 activity by CYB5, though the specific mechanism remains to be determined.

Heteromeric complex formation and crosstalk between type 1 metabotropic glutamate receptor and GABA<sub>B</sub> receptor

Heteromeric complex formation and crosstalk between type 1 metabotropic glutamate receptor and GABA<sub>B</sub> receptor

Soluble Starch Synthase Enzymes in Cereals: An Updated Review

Cereal crops have starch in their endosperm, which has provided calories to humans and livestock since the dawn of civilization to the present day. Starch is one of the important biological factors which is contributing to the yield of cereal crops. Starch is synthesized by different enzymes, but starch structure and amount are mainly determined by the activities of starch synthase enzymes (SS) with the involvement of starch branching enzymes (SBEs) and debranching enzymes (DBEs). Six classes of SSs are found in Arabidopsis and are designated as soluble SSI-V, and non-soluble granule bound starch synthase (GBSS). Soluble SSs are important for starch yield considering their role in starch biosynthesis in cereal crops, and the activities of these enzymes determine the structure of starch and the physical properties of starch granules. One of the unique characteristics of starch structure is elongated glucan chains within amylopectin, which is by SSs through interactions with other starch biosynthetic enzymes (SBEs and DBEs). Additionally, soluble SSs also have conserved domains with phosphorylation sites that may be involved in regulating starch metabolism and formation of heteromeric SS complexes. This review presents an overview of soluble SSs in cereal crops and includes their functional and structural characteristics in relation to starch synthesis.

Colorectal Cancer-Associated Smad4 R361 Hotspot Mutations Boost Wnt/β-Catenin Signaling through Enhanced Smad4-LEF1 Binding.

About 10% to 30% of patients with colorectal cancer harbor either loss of or missense mutations in SMAD4, a critical component of the TGFβ signaling pathway. The pathophysiologic function of missense mutations in Smad4 is not fully understood. They usually map to the MH2 domain, specifically to residues that are involved in heterodimeric complex formation with regulatory Smads (such as Smad2/3) and ensuing transcriptional activation. These detrimental effects suggest that SMAD4 missense mutations can be categorized as loss-of-function. However, they tend to cluster in a few hotspots, which is more consistent with them acting by a gain-of-function mechanism. In this study, we investigated the functional role of Smad4 R361 mutants by re-expressing two R361 Smad4 variants in several Smad4-null colorectal cancer cell lines. As predicted, R361 mutations disrupted Smad2/3-Smad4 heteromeric complex formation and abolished canonical TGFβ signaling. In that, they were similar to SMAD4 loss. However, RNA sequencing and subsequent RT-PCR assays revealed that Smad4mut cells acquired a gene signature associated with enhanced Lef1 protein function and increased Wnt signaling. Mechanistically, Smad4 mutant proteins retained binding to Lef1 protein and drove a commensurate increase in downstream Wnt signaling as measured by TOP/FOP luciferase assay and Wnt-dependent cell motility. Consistent with these findings, human colorectal cancers with SMAD4 missense mutations were less likely to acquire activating mutations in the key Wnt pathway gene CTNNB1 (encoding β-catenin) than colorectal cancers with truncating SMAD4 nonsense mutations. IMPLICATIONS: Our studies suggest that in colorectal cancer hotspot mutations in Smad4 confer enhanced Wnt signaling and possibly heightened sensitivity to Wnt pathway inhibitors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/823/F1.large.jpg.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, 'How are the HO-1 and P450 systems organized in the endoplasmic reticulum?' Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

Heme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum–bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected owing to their common electron donor, they generally have been studied separately. As the expressions of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function and, conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1–CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas its activity was actually stimulated at subsaturating POR. In contrast, HO-1–mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by a simple competition for POR.

Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells.

Transforming growth factor-β (TGF-β) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-β signaling can lead to breast cancer. TGF-β elicits its effects on proliferation and differentiation via specific cell surface TGF-β type I and type II receptors (i.e., TβRI and TβRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-β-induced heteromeric complex formation, activated TβRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-β acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-β signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-β-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-β-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-β receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-β-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-β-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

Crosstalk between CYP2E1 and CYP3A enzymes and its Possible Involvement in Alcohol‐Drug Interactions.

The core of the drug‐metabolizing ensemble in human liver is formed by the ensemble of different P450 enzymes co‐localized in the membrane of endoplasmic reticulum (ER). There is an emerging recognition of the fact that multiple P450 species interact with each other with the formation of heteromeric complexes, where the catalytic properties of the individual enzymes are modified. Functional consequences of these interactions are likely to be responsible for the lack of a straight correlation between the composition of the P450 ensemble and the profile of drug metabolism. Of particular importance are the changes in P450‐P450 crosstalk induced by alcohol consumption. Although the multi‐fold increase in the content of CYP2E1 in liver of alcohol consumers is well documented, the involvement of CYP2E1 in alcohol‐drug interactions is considered insignificant due to a minor role of this enzyme in drug metabolism. However, the effects of induction of CYP2E1 may also involve its effects on the functional properties of other drug‐metabolizing enzymes.In order to probe the crosstalk between CYP2E1 and other P450 species and elucidate its role in alcohol‐drug interactions we established a model of alcohol‐induced increase in CYP2E1 content that implements incorporation of purified CYP2E1 into human liver microsomes (HLM). The adopted CYP2E1 becomes a fully‐functional member of the drug‐metabolizing ensemble and interacts with other P450 enzymes with the formation of heteromeric complexes (Davydova et al. 2019, https://doi.org/10.1101/685545). Investigating the impact of CYP2E1 on CYP3A enzymes, we found that the enrichment of HLM with CYP2E1 results in an increased rate of metabolism of 7‐benzyloxyquinoline (BQ) and eliminates the homotropic cooperativity observed with this CYP3A‐specific substrate. To probe the physiological relevance of these effects we compared three pulled preparations of HLM from normal donors (N‐HLM) with a preparation obtained from heavy alcohol consumers (A‐HLM). The content of major P450 species in the samples was characterized with mass‐spectroscopic determination of their specific peptides. The content of CYP2E1 in N‐HLM samples was from 2 to 5 lower than that found in A‐HLM. In contrast, the content of CYP3A in A‐HLM was the lowest among all four preparations. Despite the lower content of CYP3A, A‐HLM exhibited much higher rate of metabolism and lower homotropic cooperativity with BQ, similar to what is observed in CYP2E1‐enriched N‐HLM. Incorporation of additional CYP2E1 into A‐HLM results in a further increase in the rate of BQ metabolism that makes it an order of magnitude higher than in the N‐HLM sample with a similar CYP3A content. Importantly, the CYP2E1‐dependent activation observed with BQ, a highly specific substrate of CYP3A, is not observable with 7‐dimethylamino‐4‐trifluoromethyl coumarin (Coumarin 152), a substrate preferentially metabolized by CYP3A, but capable of high‐affinity interactions with several other P450 species. This observation corroborates our hypothesis that the mechanisms of substrate‐dependent activation of a particular P450 enzyme through P450‐P450 crosstalk involve a reorganization of P450 heterooligomers induced by the interactions of the target enzyme with its specific substrate.Support or Funding InformationThis research was supported by the grant R21‐AA024548 from NIH.

Regions of CYP1A2 and CYP2B4 that are Involved in the Homomeric and Heteromeric Complex Formation: The Search Continues

Cytochromes P450 (CYP or P450) represent a superfamily of enzymes that interact with limited levels of NADPH‐cytochrome P450 reductase (POR) and are responsible for the oxidation of endogenous and exogenous compounds, converting them into water‐soluble products. This superfamily of heme‐containing enzymes are membrane‐bound and reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. As previously established, apart from interacting with limited levels of POR, the cytochrome P450s form homomeric and heteromeric complexes that alter the metabolic activities of the P450s involved. This study focuses on the interaction of CYP1A2 and CYP2B4. Previous work has shown that CYP1A2 is capable of forming homomeric and heteromeric complexes whereas CYP2B4 can form only heteromeric complexes. Simple binary systems containing CYP2B4 and POR exhibit typical Michaelis‐Menten saturation curves as a function of [POR], which is consistent with simple interactions between monomeric proteins. However, CYP1A2 activities for CYP1A2/POR binary systems exhibit a sigmoidal response as a function POR, which is consistent with cooperativity resulting from CYP1A2•CYP1A2 complexes. The heteromeric interaction of the two P450s causes the activity of CYP1A2 to increase and that of CYP2B4 to decrease. The objective of this study is to identify the surface regions of the proteins that interact in the CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes. Using molecular cloning we substituted protein regions of CYP1A2 with regions of CYP2B4 and vice‐versa and expressed the chimera in the HEK‐293T cells as fusion proteins with C‐terminal protein tags that were capable of bioluminescence resonance energy transfer (BRET) when the chimeras interacted. We focused on the proximal face of the P450 as a potential region for P450•P450 complex formation. The proximal face is the region close to the heme that binds to the POR when electrons are transferred to the P450s. We have divided the proximal region into three regions designated as P1, P2, and P3. The amino acids L92‐K107 constituting P1, P132‐S156 representing P2 and T438‐K451 making up the P3 region of CYP1A2 were substituted with the respective regions of CYP2B4: T92‐S107 for P1, E132‐E163 for P2 and D438‐R448 for P3. The substitution of the P2 region of CYP1A2 with the corresponding region of CYP2B4 did not affect either CYP1A2•CYP1A2 or CYP1A2•CYP2B4 complex formation as indicated by the BRET measurements. Interestingly, the substitution of the P1 region of CYP1A2 with the corresponding region of CYP2B4 into the CYP1A2 protein disrupted the CYP1A2•CYP1A2 complex. This indicates that P1 is an important region involved in CYP1A2 homomeric complex formation. The role of P1 in the CYP1A2•CYP2B4 complex formation is currently being investigated along with the role of P3 in both the CYP1A2•CYP1A2 and the CYP1A2•CYP2B4 complexes.Support or Funding InformationSupported by NIH GM123253 & ES013648

Circulating Ficolin-2 and Ficolin-3 Form Heterocomplexes.

The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.

Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms.

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation.

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa.

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.

Effects of ethanol and ethanol metabolites on the formation of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α 1 -adrenergic receptors in human vascular smooth muscle cells

Late adolescence and young adulthood, corresponding to the high school and college years, are vulnerable periods for increased alcohol and nicotine use. The dramatic increase in the prevalence of electronic cigarette use is particularly concerning in these age groups. Late adolescents and young adults are more likely to engage in cycles of binge drug consumption, and alcohol and nicotine are frequently used together. However, there are few data examining the combination of alcohol and nicotine in binge models in animal models. In this study, our objectives were to determine how voluntary nicotine consumption beginning in late adolescence influenced subsequent binge alcohol consumption in young adulthood, how a combination of alcohol and nicotine binge consumption differed from alcohol-only binge consumption, and whether nicotine would be consumed when presented in a binge procedure. Male C57BL/6J mice voluntarily consumed unsweetened alcohol and nicotine in continuous-access bottle-choice procedures in combination with cycles of drinking-in-the-dark. Our results show that experience with voluntary nicotine consumption in late adolescence did not affect subsequent binge alcohol consumption in early adulthood. However, mice that consumed nicotine in adolescence showed an initial decrease in alcohol preference, and consequently increase in nicotine preference, on the first session of combined ethanol and nicotine binge consumption in adulthood compared with mice that drank only water during late adolescence. Lastly, we found that mice readily consumed unsweetened nicotine when presented in a binge procedure, and the level of consumption exceeded the nicotine consumption observed in the combination alcohol and nicotine binge. Our data show that expansion of the patterns of alcohol and nicotine co-consumption in a mouse models is possible, which will enable us to dissect relevant molecular targets underlying these consumption patterns and better inform drug development efforts.

Investigating the Heteromerization of Metabotropic Glutamate Receptors using a Novel Single Molecule Imaging Method

Metabotropic Glutamate Receptors (mGluRs) are a type of G-Protein Coupled Receptor (GPCR) which bind glutamate as a neurotransmitter and are found in neurons throughout the brain and peripheral tissues. mGluRs are known to function as stable homodimers, but recent evidence suggests that the formation of heteromeric complexes may also occur. Knowledge of the stoichiometry of these complexes in vivo is crucial for the development of pharmacological compounds designed to act on them. Here we investigate the complexes formed by mGluRs using various optical methods including Single Protein Recovery After Dilution (SPReAD), a novel single molecule imaging technique we have developed which utilizes VSV-G mediated cell fusion in order to dilute the concentration of fluorescently tagged molecules of interest. This enables in vivo imaging without introducing harsh detergents or altering the level of expression.

Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37°C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.

Toward a systems approach to the human cytochrome P450 ensemble: interactions between CYP2D6 and CYP2E1 and their functional consequences

Functional cross-talk among human drug-metabolizing cytochrome P450 through their association is a topic of emerging importance. Here, we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochrome P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by 3,4-methylenedioxymethylamphetamine, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that preincubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 hetero-oligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.