Transfected HEK293T Cells Research Articles

The AL626 antibody recognizes an ALFA-tagged recombinant protein by immuno-electron microscopy

The AL626 antibody against the ALFA tag recognizes an ALFA-tagged human CD1b protein by immuno-electron microscopy in transfected HEK293T cells.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

CD20-Modified Exosomes Delivered E260 Inhibits Progression of Diffuse Large B-Cell Lymphoma By Restoring Hippo Activity

Introduction: Fer tyrosine kinase (Fer) is ubiquitously expressed in various cancer types and has been proved to play a pivotal role in tumorigenesis and metastasis. E260, as an effective inhibitor of Fer, is selective lethal to cancer cells. However, the effect of Fer and E260 in diffuse large B-cell lymphoma (DLBCL) remain elusive. Thus, we set out to investigate the function and mechanisms of E260 targeting Fer in DLBCL for a treatment purpose. Methods: The impact of Fer expression to predict clinical outcome was evaluated in 50 newly diagnosed DLBCL and 30 healthy donors between 2014 and 2019 by immunohistochemistry. This study was approved by the ethics board of Shandong Provincial Hospital, and informed consent was obtained. Next, we assessed the oncogenic effect of Fer by using gain-and loss-of-function assays. Fer inhibitor E260 was used in the treatment of DLBCL cells. CD20 targeted exosomes were isolated from CD20 transfected HEK293T cells and then incubated with E260 in order to generate CD20-EXO-E260. The anti-tumor efficacy of E260 and CD20-EXO-E260 were assessed by cell proliferation, apoptosis, migration and drug sensitivity assays. To elucidate the regulatory mechanism, we test the expression of Hippo-YAP pathway and YAP location after E260 treated by western blotting and immunofluorescent staining. Interaction of the Fer and AJUBA was further validated by coimmunoprecipitation (Co-IP) assay. To further explore the clinical value of Fer, we isolated and test Fer expression in exosomes from the plasma of DLBCL patients. ROC curve was performed to test the diagnostic value of exosomal Fer in DLBCL. Results: To explore the association of Fer expression with DLBCL tumorigenesis, we examined Fer expression using immunohistochemical staining. Fer was upregulated in DLBCL patients (Fig.1A) and correlated with adverse clinical parameters (Fig.1B) and shorter overall survival time (Fig.1C). Since Fer was also increased in the DLBCL cell lines, we then set out to explore the biological function of Fer in DLBCL cells (Fig.1D). Transfected efficacy was verified by western blot (Fig2.A). In vitro gain-and loss-of-function assays demonstrated the stimulatory role of Fer on cell viability, migration and chemoresistance (Fig2.B-D). Recently, exosomes have been emerged as promising drug delivery carriers. Thus, we generated CD20-modified exosomes as an alternative to targeting deliver E260 to DLBCL cells. This event not only facilitated the targeting efficacy of E260 (Fig.3A) but also showed higher efficacy than E260 in eliminating DLBCL progression through inhibiting cell proliferation and chemotaxis, enhancing cell apoptosis and drug sensitivity both in vitro and in vivo (Fig.3B-F). In the view of GO and KEGG analysis, Fer mainly enrichment in regulating cell proliferation, adhesion, response to drug and Hippo pathway in cancer (Fig.4A). Consistent with this, we demonstrated that E260 activated Hippo pathway and leading to YAP cytoplasmic sequestration (Fig.4B-C). Co-IP assay confirmed the physical interaction between Fer and AJUBA. Furthermore, upregulated AJUBA reversed E260 mediated cell proliferation inhibition (Fig.4D). Thus, we suggest that E260 inhibits DLBCL progression by downregulating AJUBA which restores Hippo tumor-suppressor activity. In the mice model, the levels of plasma exosomal Fer could reflex the tumor burden (Fig.5A). We next detected that exosomal Fer and AJUBA expression increased in the plasma of DLBCL patients (Fig.5B-C) and released after R-CHOP therapy (Fig.5D). Besides, Fer expression was positively related with AJUBA (Fig.5E). As shown in the Figure 5F-G, exosomal Fer could act as a promising indicator in early diagnosis and disease progression for DLBCL patients. Conclusion: We demonstrated for the first time the oncogenic role of Fer in DLBCL progression and therefore provided an appealing therapeutic target. CD20-targeted exosomes delivery E260 are more effective in eliminating DLBCL progression through diminishing AJUBA and activating Hippo pathway. Furthermore, we provided circulating exosomal Fer as a novel non-invasive biomarker for DLBCL diagnosis. Disclosures No relevant conflicts of interest to declare.

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

BackgroundCirculating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium‐derived prostacyclin and nitric oxide stimulate cAMP‐ and cGMP‐dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. ObjectivesWe wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide‐dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. MethodsPhosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos‐tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull‐down assays were performed to analyze protein interaction and function. ResultsOur data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA‐specific GTPase‐activating protein Myo9b and the guanine nucleotide exchange factor GEF‐H1. We identify Myo9b S1354 and guanine nucleotide exchange factor‐H1 (GEF‐H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA‐GTP levels. GEF‐H1 S886 phosphorylation stimulates binding of 14‐3‐3β and has been shown to inhibit GEF function by facilitating binding of GEF‐H1 to microtubules. Microtubule disruption increases RhoA‐GTP levels confirming the importance of GEF‐H1 in platelets. ConclusionPhosphorylation of RhoA regulatory proteins Myo9b and GEF‐H1, but not RhoA itself, is involved in cyclic nucleotide‐mediated control of RhoA in human platelets.

SMIM1 missense mutations exert their effect on wild type Vel expression late in erythroid differentiation.

Vel expression on erythrocytes is variable due to polymorphisms, complicating Vel typing. Weak Vel expression can be caused by mutations within SMIM1 in a heterozygous setting, suggesting a dominant negative effect of SMIM1 mutants on wild type (wt)SMIM1 expression. Here we report how SMIM1 expression is regulated during erythropoiesis, to understand its variable expression on erythrocytes. Peripheral blood reticulocytes at different stages, cultured erythroid precursors and HEK293T cells were used to investigate expression and putative competition between wtSMIM1 and mutated SMIM1 VEL*01W.01, (c.152T>A (p.Met51Lys)), VEL*01W.02 (c.152T>G (p.Met51Arg)), and VEL*01W.03 (c.161T>C (p.Leu54Pro)). Depending on the mutations in SMIM1 an effect on total and membrane expression of SMIM1 was observed in transfected HEK293T cells, but co-expression of wtSMIM1 and mutatedSMIM1 did not have an effect on wtSMIM1 membrane expression. During differentiation of donors expressing VEL*01W.01, VEL*01W.03, Vel-positive, Vel-negative (homozygote SMIM1*64_80del), and Vel-heterozygote SMIM1*64_80del primary human erythroblasts no overt defect was found in Vel expression dynamics or total SMIM1 expression levels when compared with wtSMIM1 erythroblasts. However, during enucleation, total Vel expression was significantly lower on reticulocytes of Vel-weak donors expressing heterozygote mutated SMIM1 compared to Vel-positive or Vel-heterozygote SMIM1*64_80del donors, while Vel expression on extruded nuclei was maintained. In addition, reticulocyte maturation in vivo showed further loss of Vel expression in these individuals and nearly absent on erythrocytes. These results suggest that SMIM1 mutations exert a dominant negative effect on wtSMIM1 probably by affecting SMIM1 multimerization and thereby Vel epitope presentation at the latest stages of erythroid differentiation.

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live cells using the bioluminescence resonance energy transfer technique.

As of today, only acute effects of RF fields have been confirmed to represent a potential health hazard and they are attributed to non-specific heating (≥ 1°C) under high-level exposure. Yet, the possibility that environmental RF impact living matter in the absence of temperature elevation needs further investigation. Since HSF1 is both a thermosensor and the master regulator of heat-shock stress response in eukaryotes, it remains to assess HSF1 activation in live cells under exposure to low-level RF signals. We thus measured basal, temperature-induced, and chemically induced HSF1 trimerization, a mandatory step on the cascade of HSF1 activation, under RF exposure to continuous wave (CW), Global System for Mobile (GSM), and Wi-Fi-modulated 1800MHz signals, using a bioluminescence resonance energy transfer technique (BRET) probe. Our results show that, as expected, HSF1 is heat-activated by acute exposure of transiently transfected HEK293T cells to a CW RF field at a specific absorption rate of 24W/kg for 30min. However, we found no evidence of HSF1 activation under the same RF exposure condition when the cell culture medium temperature was fixed. We also found no experimental evidence that, at a fixed temperature, chronic RF exposure for 24h at a SAR of 1.5 and 6W/kg altered the potency or the maximal capability of the proteasome inhibitor MG132 to activate HSF1, whatever signal used. We only found that RF exposure to CW signals (1.5 and 6W/kg) and GSM signals (1.5W/kg) for 24h marginally decreased basal HSF1 activity.

Production of Chimeric Hepatitis B Virus Surface Antigens in Mammalian Cells.

The small (S) envelope protein of the Hepatitis B Virus (HBV), HBV-S, has the unique ability to self-assemble into highly immunogenic subviral particles (SVPs), in the absence of other viral factors, in eukaryotic cells, including those of nonhepatic origin. This feature is currently exploited for generation of SVPs exposing heterologous epitopes on their surface that can be used as vaccine candidates to target various diseases. Here, we describe a simple and robust method for production of such chimeric HBV-S protein-based SVPs in transiently transfected HEK293T cells and purification from cell supernatants by ultracentrifugation on sucrose cushion and sucrose step gradients. The SVPs obtained by this methodology have been successfully used in immunogenicity studies in animal models.

Secreted gingipains from Porphyromonas gingivalis induce microglia migration through endosomal signaling by protease-activated receptor 2

Much attention has been paid to the connection between periodontitis and Alzheimer's disease (AD). We previously showed that infection of P. gingivalis, one of major periodontal pathogen causing periodontitis, induced migration and inflammatory responses in murine microglia through gingipain-induced activation of protease-activated receptor 2 (PAR2). In this study, we have attempted to clarify effects of secreted gingipains on cell migration of human microglial cell line, cleavage sites of PAR2 by gingipains and subsequent signaling pathways. P. gingivalis culture supernatant induced migration and membrane ruffling, which is necessary for microglia migration, in human microglial cell line HMC3 cells through PAR2. These effects were mainly mediated by gingipains, because cell migration and membrane ruffling were dramatically inhibited by treatment with gingipain inhibitors. Furthermore, pharmacological and genetic inhibition of Src kinase and β-arrestin, which are important for the internalization of G protein-coupled receptors, significantly inhibited P. gingivavlis culture supernatant-induced membrane ruffling in HMC3 cells. After treatment with P. gingivalis culture supernatant in Flag-PAR2-HA transfected HEK293T cells, Flag was removed from the cell surface, and HA was detected in the cytosol, indicating the internalization of PAR2. Furthermore, the phosphorylation level of ERK1/2 increased in PAR2-transfected HEK293T cells after treatment with P. gingivalis culture supernatant. The gingipain inhibitors, Src kinase inhibitor and β-arrestin knockdown suppressed PAR2 internalization and ERK1/2 phosphorylation. These observations suggest that secreted gingipains from P. gingivalis induce Src- and β-arrestin-dependent internalization of PAR2 and further activate the ERK1/2 pathway to promote migration of microglia. PAR2 are activated by the tethered ligands exposed by cleavage of extracellular N-terminal of PAR2. We also estimated potential gingipain cleavage sites in PAR2 and exposed tethered ligands, which are required for PAR2 internalization and membrane ruffling. The identified mechanism in this study might contribute to the retrogression of sporadic AD in patients after infection with P. gingivalis.

HIV-1 Nef promotes ubiquitination and proteasomal degradation of p53 tumor suppressor protein by using E6AP

Human Immunodeficiency Virus-1 (HIV-1) Nef promotes p53 protein degradation to protect HIV-1 infected cells from p53 induced apoptosis. We found that Nef mediated p53 degradation is accomplished through ubiquitin proteasome pathway in an Mdm2-independent manner. By GST pulldown and immunoprecipitation assays, we have shown that Nef interacts with E3 ubiquitin ligase E6AP in both Nef transfected HEK-293T cells and HIV-1 infected MOLT3 cells. The p53 ubiquitination and degradation was found to be enhanced by Nef with E6AP but not by Nef with E6AP-C843A, a dominant negative E6AP mutant. We show that Nef binds with E6AP and promotes E6AP dependent p53 ubiquitination. Further, Nef inhibits apoptosis of p53 null H1299 cells after exogenous expression of p53 protein. The p53 dependent apoptosis of H1299 cells was further reduced after the expression of Nef with E6AP. However, Nef mediated reduction in p53 induced apoptosis of H1299 cells was restored when Nef was co-expressed with E6AP-C843A. Thus, Nef and E6AP co-operate to promote p53 ubiquitination and degradation in order to suppress p53 dependent apoptosis. CHME3 cells, which are a natural host of HIV-1, also show p53 ubiquitination and degradation by Nef and E6AP. These results establish that Nef induces p53 degradation via cellular E3 ligase E6AP to inhibit apoptosis during HIV-1 infection.

TRPV6 variants confer susceptibility to chronic pancreatitis in the Chinese population.

Chronic pancreatitis (CP) is a progressive fibroinflammatory syndrome of the pancreatic tissue caused by genetic and environmental factors. Previously reported susceptibility genes in CP explain less than half of the apparent heritability. To uncover novel pathogenic mechanisms, we initially performed low-coverage whole-genome sequencing on 464 Chinese CP patients and 504 controls. The transient receptor potential cation channel, Subfamily V, Member 6 (TRPV6) gene was found to be significantly associated with CP after a burden test of aggregated rare nonsynonymous variants with a combined annotation dependent depletion score > 20 (p = .020). In the replication stage, we analyzed the entire coding sequence and exon/intron boundaries of the TPRV6 gene by Sanger sequencing in another 205 patients with CP and 105 controls. Integration of the findings from the two stages resulted in the identification of 25 TRPV6 variants: 1 rare nonsense variant, 20 rare missense variants, and 4 common missense variants. Loss-of-function variants, as determined by intracellular Ca2+ concentration in transfected HEK293T cells, were significantly overrepresented in patients as compared to controls (9/669 [1.35%] vs. 1/609 [0.16%]; odds ratio = 8.29; p = .022). This study provides evidence suggesting that TRPV6 is a novel susceptibility gene for CP.

β-Arrestin-2-Dependent Mechanism of GPR52 Signaling in Frontal Cortical Neurons.

The orphan Gαs-coupled receptor GPR52 is expressed exclusively in the brain, predominantly in circuitry relating to symptoms of neuropsychiatric and cognitive disorders such as schizophrenia. While GPR52 agonists have displayed antipsychotic and procognitive efficacy in murine models, there remains limited evidence delineating the molecular mechanisms of these effects. Indeed, previous studies have solely reported canonical cAMP signaling and CREB phosphorylation downstream of GPR52 activation. In the present study, we demonstrated that the synthetic GPR52 agonist, 3-BTBZ, equipotently induces cAMP accumulation, ERK1/2 phosphorylation, and β-arrestin-1 and -2 recruitment in transfected HEK293T cells. In cultured frontal cortical neurons, however, 3-BTBZ-induced ERK1/2 phosphorylation was significantly more potent than cAMP signaling, with a more prolonged signaling profile than that in HEK293T cells. Furthermore, knock down of β-arrestin-2 in frontal cortical neurons abolished 3-BTBZ-induced ERK1/2 phosphorylation, but not cAMP accumulation. These results suggest a β-arrestin-2-dependent mechanism for GPR52-mediated ERK1/2 signaling, which may link to cognitive function in vivo. Finally, these findings highlight the context-dependence of GPCR signaling in recombinant cells and neurons, offering new insights into translationally relevant GPR52 signaling mechanisms.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BackgroundHypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition.MethodsWe inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT.ResultsHypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and β-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and β-catenin and protein level of β-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of β-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant.ConclusionbHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Construction of a Novel, Dual, Self-Replicating Minicircle; an Efficient Tool in Transdifferentiation

Background & Aim Minicircles (MCs) are circular DNAs contain minimal eukaryotic expression cassette (EEC) devoid of bacterial backbone (BB). Due to low-toxicity and Immunogenicity of Minicircles, these vectors could serve as an attractive alternative in therapeutic applications such as DNA vaccines, pluripotent cells or gene-based therapies. The inclusion of scaffold/matrix attachment region (S/MAR) elements in minicircles can increase long-term expression of transgene in the dividing cells. In this study we constructed a dual reporter minicircle (DR-MC) containing an S/MAR element and two different reporter genes, mCherry and EGFP, which express in different stages after transfection into eukaryotic cells. First reporter gene is flanked by two Lox recognition sites and after adequate level of expression removes by the intramolecular Cre-madiated recombination. So, the second reporter locate adjacent to promoter and express. Reporter genes could be replaced by the gene of interests, involving in transdifferentiation. Methods, Results & Conclusion Two different fragments were amplified by PCR, loxP-mCherry-BGHpA-loxH (LML) and EGFP-SMAR-SV40pA-SV40promoter (ESS), which respectively were cloned between attB-attP sites of parental plasmid (PP-LML-ESS). Minicircle producing bacteria (ZYCY10P3S2T) were transformed with PP-LML-ESS, producing DR-MCs after induction by L-Arabinose. DR-MCs were transfected into HEK293T cells and mCherry expression was assessed by fluorescent microscopy and Flow cytometry. Tat-NLS-Cre recombinase was added to transfected cell's culture medium and EGFP expression was measured in several time points. Reporter-expressing cells were picked up to generate stable cell lines, evaluated by qRT-PCR and FISH analysis to determine the episomal state of MCs and copy numbers. The accuracy of constructed PP-LML-ESS and DR-MC was determined by restriction digestion. mCherry expression in transfected HEK293T cells confirmed the elimination of BB. Tat-NLS-Cre recombinase led to mCherry excision and EGFP expression. By tracking reporter-expressing cell lines after several passages we realized that DR-MC could replicate episomaly with maintained copy numbers. In conclusion DR-MCs propose safe, long-term and efficient systems which could carry several genes with adjustable expression time points. These characteristics enable DR-MCs to be used in cell's lineage reprogramming, in which one mature somatic cell transforms into another mature somatic cell with different gene expression profile.

Constitutive activity of dopamine receptor type 1 (D1R) increases CaV2.2 currents in PFC neurons.

Alterations in dopamine receptor type 1 (D1R) density are associated with cognitive deficits of aging and schizophrenia. In the prefrontal cortex (PFC), D1R plays a critical role in the regulation of working memory, which is impaired in these cognitive deficit states, but the cellular events triggered by changes in D1R expression remain unknown. A previous report demonstrated that interaction between voltage-gated calcium channel type 2.2 (CaV2.2) and D1R stimulates CaV2.2 postsynaptic surface location in medial PFC pyramidal neurons. Here, we show that in addition to the occurrence of the physical receptor-channel interaction, constitutive D1R activity mediates up-regulation of functional CaV2.2 surface density. We performed patch-clamp experiments on transfected HEK293T cells and wild-type C57BL/6 mouse brain slices, as well as imaging experiments and cAMP measurements. We found that D1R coexpression led to ∼60% increase in CaV2.2 currents in HEK293T cells. This effect was occluded by preincubation with a D1/D5R inverse agonist, chlorpromazine, and by replacing D1R with a D1R mutant lacking constitutive activity. Moreover, D1R-induced increase in CaV2.2 currents required basally active Gs protein, as well as D1R-CaV2.2 interaction. In mice, intraperitoneal administration of chlorpromazine reduced native CaV currents' sensitivity to ω-conotoxin-GVIA and their size by ∼49% in layer V/VI pyramidal neurons from medial PFC, indicating a selective effect on CaV2.2. Additionally, we found that reducing D1/D5R constitutive activity correlates with a decrease in the agonist-induced D1/D5R inhibitory effect on native CaV currents. Our results could be interpreted as a stimulatory effect of D1R constitutive activity on the number of CaV2.2 channels available for dopamine-mediated modulation. Our results contribute to the understanding of the physiological role of D1R constitutive activity and may explain the noncanonical postsynaptic distribution of functional CaV2.2 in PFC neurons.

Novel Mutations in OCTN2 Identified in Newborn Screening Exhibit Reduced Function and Lead to Carnitine Transporter Deficiency

Newborn screening programs have revolutionized the diagnosis and treatment of genetic disorders. A compelling example is found in SLC22A5 encoding OCTN2, a bi‐directional plasma membrane transporter, which acts in the influx carnitine and efflux of drugs. Newborns with low plasma carnitine levels and biallelic SLC22A5 variants are diagnosed with Carnitine Transporter Deficiency (CTD), a potentially fatal disorder that can be treated with L‐carnitine supplementation. Here, we sought to validate the role of novel OCTN2 variants in CTD via functional characterization of mutant transporters in vitro.In a recent study (PMID 31364285), three novel OCTN2 missense variants were identified in newborn screening. Following site‐directed mutagenesis and transient transfection in HEK293T cells, we performed isotopic uptake assays using 14C‐carnitine. Sodium phenylbutyrate, a chemical chaperone previously shown to increase function of other OCTN2 variants, was used to treat transfected cells. Welch’s two‐sample t‐test was used to determine significance.Two novel missense variants, p.Asp165Glu and p.Gly473Val, exhibited substantial reduction of carnitine transport, retaining less than 5% of wildtype transporter function (p = 9.63*10−6 and 1.02*10−5, respectively). In contrast, only moderate reduction was observed for p.Met433Thr, which retained 61% wildtype function (p = 0.021). Sodium phenylbutyrate showed trends (which were not significant) toward increasing the function of all three variants, suggesting that intracellular errors of protein folding, processing, or trafficking may contribute to their reduced function.Our studies validate the role of three novel variants in CTD, none of which have been associated previously with the disorder. The studies are consistent with the idea that even partial loss‐of‐function alleles may be disease‐causing for CTD. Finally, we suggest that sodium phenylbutyrate represents a potential modulator of OCTN2 function, with further studies warranted to confirm effects on function and membrane localization of OCTN2 variants.Support or Funding InformationThis work is supported by the Chan Zuckerberg Biohub.

Kinetensin is an Endogenous β‐arrestin‐biased Ligand of Angiotensin AT1 receptor

Kinetensin, a nonapeptide having similarities to neurotensin and angiotensin II has been isolated from human plasma more than 30 years ago, but its patho‐physiological functions and signal transduction mechanisms remain elusive. In this work, we investigated the effects of kinetensin on G protein‐coupled receptors (GPCRs), one of the largest families of the human genome and the biological target of many peptides. Using Presto‐tango system, the effects of this peptide were tested on a panel of 178 class A GPCRs. Kinetensin (10−6M) stimulated β‐arrestin activation in HTLA cells transfected with angiotensin type 1 receptor (AT1R) by 638±45% compared to basal levels (n=16). At the same concentration, kinetensin also stimulated β‐arrestin activation by histamine H1 receptor (201+22%), melatonin MT1 (187±12%) and MT2 (183±7%) receptors, MRGPRX3 (179±14%), GPR83 (186±10%) and GPR88 (190±10% n=3 in each case), but these effects were significantly smaller compared to the effects on AT1R. In contrast, kinetensin had no significant effect on β‐arrestin activation in cells transfected with other GPCRs, including receptors related to renin‐angiotensin system like AT2, MAS1 and MAS1L, or with neurotensin receptors (NTS1 and NTS2). The effects of the peptide on AT1R‐stimulated β‐arrestin activation were reproduced in HEK293T cells using nanoBRET method. These experiments demonstrated that maximal effects of kinetensin at 10−6M were 39±8% from the effects of angiotensin II (angII, 10−6M), with an EC50 of 115+21 nM (n=4). We also tested the effects of kinetensin on [Ca2+]i (a marker of G‐protein activation) in AT1R transfected HEK293T cells and we found that its effects were only a fraction of the effects of angII (14±8%, n=4). These data indicate that kinetensin is a β‐arrestin biased agonist at AT1R. Interestingly, angiotensin 1‐7 (ang1‐7), a heptapeptide with opposite actions to angII and favorable effects on the regulation of cardio‐vascular activities, demonstrated comparable effects in cells transfected with AT1R (21+7% stimulation of β‐arrestin activation and 4±8% increases in [Ca2+]i at 10−6 M, n=4). In this respect, kinetensin also resembles the effects of novel AT1R β‐arrestin biased agonist TRV027, which is currently tested in clinical trials for the treatment of acute heart failure. Overall, our results show that the nonapeptide kinetensin differentially modulates AT1R‐induced β‐arrestin and G‐protein activation, and these effects can be relevant to target this GPCR in cardio‐vascular diseases.Support or Funding InformationThis work was supported in part by grants from National Science Foundation (Nos. 1708959 & 1924092) and Office of Naval Research (Nos. N00014‐18‐1‐2145 & N00014‐19‐1‐2602) to YF, U.S. Department of Veterans Affairs (I01 BX004294 to CMF and EL) and National Institutes of Health (HL093178 and COBRE P30GM106392), and LSUHSC‐NO Research Enhancement Program to EL.

Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor-Receptor Interaction in a Model of Movement Disorders.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic–cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic–cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R–M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R–M1R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.

Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling.

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post-synaptic neurons "on demand" and released to bind pre-synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2-AG), is primarily synthesized by diacylglycerol lipase-α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post-synaptic depolarization and/or the activation of Gq -coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2-AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP-dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho-Ser798-specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2-AG mobilization in mouse striatal slices by manipulating D1-dopamine receptor (D1R) signaling and assessing depolarization-induced suppression of excitation, a DGLα- and 2-AG-dependent form of short-term synaptic depression. The magnitude of depolarization-enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre-incubation with a D1R agonist, and this enhancement was blocked by post-synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling.