Decreased Cell Surface Expression Research Articles

Identification of a novel Scn3b mutation in a Chinese Brugada syndrome pedigree: implications for Nav1.5 electrophysiological properties and intracellular distribution of Nav1.5 and Navβ3.

The Scn3b gene encodes for Navβ3, a pivotal regulatory subunit of the fast sodium channel in cardiomyocytes. However, its mutation status in the Chinese population suffering from Brugada Syndrome (BrS) has not been characterized, and the contributory pathophysiological mechanisms to disease pathology remain undefined. A Scn3b (c.260C>T, p.P87l) mutation was identified in a patient with BrS of Chinese descent. Functional analyses demonstrated that sodium channel activation for the wild type, mutant samples, and co-expression of both commenced at -55 mv and peaked at -25 mv. The mutant group exhibited a notable reduction, approximately 60%, in peak sodium channel activation current (INa) at -25 mv. The parameters for half-maximal activation voltages (V1/2) and slope factors (k) showed no significant differences when comparing wild type, mutant, and combined expression groups (P = 0.98 and P = 0.65, respectively). Additionally, no significant disparities were evident in terms of the steady-state sodium channel inactivation parameters V1/2 and k (with P-values of 0.85 and 0.25, respectively), nor were there significant differences in the activation time constant τ (P = 0.59) and late sodium current density (P = 0.23) across the wild-type, mutant, and co-expressed groups. Confocal imaging and Western blot analysis demonstrated decreased plasma membrane localization of SCN3B and SCN5A in the P87l group. Computational simulations of cardiac action potentials suggested that SCN3B P87l can alter the morphology of the action potentials within the endocardium and epicardium while reducing the peak of depolarization. The pathogenic impact of the Scn3b P87l mutation predominantly originates from a reduction in peak INa activation current coupled with decreased cell surface expression of Nav1.5 and Navβ3. These alterations may influence cardiac action potential configurations and contribute to the risk of ventricular arrhythmias in individuals with BrS.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

SORLA‐retromer complex formation rely on receptor dimerization within the endosome

AbstractBackgroundSORLA acts as an endosome recycling receptor together with the retromer complex to escort cargo through the endo‐lysosomal tubular network. SORLA is encoded by the SORL1 gene, which is the gene most frequently mutated in patients with Alzheimer’s disease, although it is still not clear which of the many variants are (the most) pathogenic. Here, we studied the biochemistry and cell biology of a SORL1 variant that is found in several European families.MethodsWe used transfected cells to study SORLA maturation and shedding of the soluble sSORLA by Western blotting. Expression of non‐mutated and mutated receptor at the cell surface was determined by flow cytometry, and the intracellular localization and overlap with markers of Endoplasmic reticulum and endosome compartments investigated by immunocytochemistry and confocal microscopy. The cellular distribution of receptor dimerization was visualized using bimolecular fluorescence complementation and proximity ligation assays, and quantified using the GFP‐trap method.ResultsWe showed that non‐mutated SORLA forms a dimer in retromer‐coated endosome structures, while the pathogenic SORLA protein is unable to form dimers, although it traffics to the endosome. This lack of dimerization in the endosome correlates with strongly decreased receptor maturation, expression at the cell surface, and shedding of its luminal sSORLA domain.ConclusionSORLA dimer formation is required for its association with retromer, which is important for efficient receptor maturation and delivery to the cell surface. Decreased cell surface expression of mutated protein with impaired dimerization leads to less shedding, showing how maturation and sSORLA production can reliably inform on the pathogenicity of SORL1 genetic variants in Alzheimer’s disease.

Abstract 1287: DEC2, a circadian rhythm transcription factor, promotes growth, metastasis and dormancy by facilitating immune cell invasion through decreasing cell surface expression of MHC-1 in a novel model of pancreatic cancer dormancy

Abstract Latent recurrence following curative-intent pancreatic cancer surgery is a major clinical problem thought to be due to the reactivation of dormant tumor cells that disseminate before the primary tumor has been removed. How dormancy is established and ultimately reversed to drive recurrence is poorly understood. Here we introduce a novel mouse model of pancreatic cancer dormancy in which we establish distal pancreatic tumors by orthotopic injection, resect them four weeks later and then follow the mice for recurrence. We observed recurrence patterns and survival outcomes that mimic human patients undergoing surgery for pancreatic cancer where two thirds of the mice succumb to early metastatic recurrence (median survival 26 days) and one third of the mice (called dormant mice) live substantially long (median survival 554 days) without clinic evidence of disease yet harbor disseminated tumor cells in most organs of the body. Disseminated tumor cells isolated from the livers of dormant mice were quiescent, exhibited stem cell properties and upregulated the expression of Dec2, a circadian rhythm gene. Overexpression of Dec2 induced quiescence suggesting that it may be a driver of the dormant phenotype. Curiously, endogenous Dec2 actually increased the growth and metastasis of nondormant tumors. Dec2WT and Dec2 knockout cell lines were used to generate resectable orthotopic tumors in immune-competent mice, and loss of Dec2 decreased tumor growth, dissemination and metastasis, and the median survival of mice with DecWT tumors was 25 days whereas 70% of the mice with the otherwise isogenic Dec2KOs were still alive after 200 days. Notably, these differences in tumor size, dissemination, metastasis and survival were abrogated in nude mice, indicating an immune mediated mechanism. Dec2WT and Dec2KO cell lines expressed the same total amount of the antigen-presenting protein MHC-1, but much more of the MHC-1 was present on the surface of Dec2KO cell lines than on the Dec2WT cell lines. MHC-1 requires peptide loading before transport to the cell surface, and we see evidence for reduced production of small peptides in the DecKO cells. For instance the half-life of luciferase, a very potent immunogen in mice, was reduced in the Dec2KO cells, potentially providing small peptides for MHC-1 biosynthesis. Protein turnover may be upregulated to compensate for increases in translation, as ribosomal genes are more highly expressed in Dec2KO cells, including many c-Myc pathway genes. Indeed, Western blot analysis revealed upregulation of c-Myc protein in Dec2KO cells. These data suggest that Dec2 plays a novel role in the immune system by decreasing MHC-1 cell surface expression through decreasing peptide generation, protein translation and turnover. Citation Format: Chris R. Harris, Crissy Dudgeon, Orjola Prela, Lan Wang, Anthony Casabianca, Juliana Cazarin de Menezes, Christina Davidson, Paula Vertino, Brian Altman, Darren Carpizo. DEC2, a circadian rhythm transcription factor, promotes growth, metastasis and dormancy by facilitating immune cell invasion through decreasing cell surface expression of MHC-1 in a novel model of pancreatic cancer dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1287.

Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Abstract 9808: Deletion of Arrdc4 Enhances Myocardial Glucose Transport and Ameliorates Ischemic Injury

Cardiomyocytes depend on glycolysis instead of oxidative phosphorylation during hypoxia and utilize glucose as a primary anaerobic energy source. Cardiac glucose transport is facilitated by the expression of glucose transporters (GLUT1 and GLUT4) on the cell surface. The arrestins are a family of proteins that regulate the signaling and trafficking of different receptors and transporters on the plasma membrane. As a scaffold protein, beta-arrestins mediate endocytosis and signal termination of beta-adrenergic receptors in cardiomyocytes. However, unlike the well-characterized beta-arrestins, the physiological roles of the related proteins termed alpha-arrestins remain largely unknown in the heart. In this study, we discovered for the first time that a member of alpha-arrestins, Arrestin Domain-Containing Protein 4 (Arrdc4), functions as a regulatory scaffold protein of GLUT1 and controls cardiac glucose metabolism. We found that Arrdc4 interacts with GLUT1 and promotes its endocytosis in cardiomyocytes. Interestingly, the cardiac expression of Arrdc4 is significantly upregulated by myocardial ischemia. Arrdc4 decreases cell surface expression of GLUT1 and suppresses glucose transport in the wild-type (WT) mouse heart. Then, we generated a novel Arrdc4 knockout (KO) mouse using CRISPR/Cas9 genome editing and characterized the effects of Arrdc4 in an animal model of myocardial infarction (MI) by coronary artery ligation in vivo. Surprisingly, Arrdc4-KO mice exhibit smaller infarct size, fewer apoptotic cells, and higher left ventricular (LV) fractional shortening, resulting in better post-MI survival than WT mice. Furthermore, using the Langendorff perfusion system, we found that Arrdc4-KO hearts augment cardiac glucose transport and preserve better LV mechanical function during anaerobic perfusion than WT hearts ex vivo. These results demonstrate that Arrdc4-KO cardiomyocytes coordinately maintain the optimal expression of GLUT1 on the cell surface, which leads to a metabolic rearrangement to secure energy homeostasis under oxygen- and nutrient-limited environments. In conclusion, we identified Arrdc4 as a new adaptor protein for GLUT1 in cardiomyocytes and a key player in cardiac metabolism in the ischemic heart.

TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory periodic fever syndrome associated with heterozygous mutations in TNFRSF1A, which encodes TNF receptor type I (TNFR1). Although possible proinflammatory mechanisms have been proposed, most previous studies were performed using in vitro overexpression models, which could lead to undesirable inflammatory responses due to artificial overexpression. It is crucial to reproduce heterozygous mutations at physiological expression levels; however, such studies remain limited. In this study, we generated TRAPS mutant mice and analyzed their phenotypes. Three Tnfrsf1a mutant strains were generated by introducing T79M, G87V, or T90I mutation. T79M is a known mutation responsible for TRAPS, whereas G87V is a TRAPS mutation that we have reported, and T90I is a variant of unknown significance. Using these murine models, we investigated whether TRAPS mutations could affect the inflammatory responses in vivo and in vitro. We found that none of the mutant mice exhibited detectable inflammatory phenotypes under standard housing conditions for 1 year. Interestingly, TRAPS mutant (T79M and G87V) mice had reduced mortality rates after the administration of lipopolysaccharide (LPS) and D-galactosamine, which induce TNFα-dependent lethal hepatitis. Moreover, TRAPS mutations strongly suppressed the development of TNFα-mediated arthritis when crossed with human TNFα transgenic mice. In in vitro primary bone marrow-derived macrophage cultures, the T79M and G87V mutations attenuated the inflammatory responses to TNFα compared with the wild-type, whereas these mutations did not alter the responsiveness of these cells to LPS. The T90I mutant macrophages behaved similarly to wild type in response to LPS and TNFα. The TNFR1 levels were increased in whole-cell lysates of TRAPS mutant macrophages, whereas the cell surface expression of TNFR1 was significantly decreased in TRAPS mutant macrophages. Taken together, TRAPS mutations did not augment the inflammatory responses to TNFα and LPS; instead, they suppressed the response to TNFα via decreased cell surface expression of TNFR1. The stimulation of lymphotoxin-α, adenosine triphosphate, and norepinephrine in primary macrophages or various stimuli in murine splenocytes did not induce detectable inflammatory responses. In conclusion, TRAPS mutations suppressed responsiveness to TNFα, and TRAPS-associated inflammation is likely induced by unconfirmed disease-specific proinflammatory factors.

CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development.

Hematopoietic stem cells (HSCs) express a large variety of cell surface receptors that are associated with acquisition of self-renewal and multipotent properties. Correct expression of these receptors depends on a delicate balance between cell surface trafficking, recycling, and degradation and is controlled by the microtubule network and Golgi apparatus, whose roles have hardly been explored during embryonic/fetal hematopoiesis. Here we show that, in the absence of CLASP2, a microtubule-associated protein, the overall production of HSCs is reduced, and the produced HSCs fail to self-renew and maintain their stemness throughout mouse and zebrafish development. This phenotype can be attributed to decreased cell surface expression of the hematopoietic receptor c-Kit, which originates from increased lysosomal degradation in combination with a reduction in trafficking to the plasma membrane. A dysfunctional Golgi apparatus in CLASP2-deficient HSCs seems to be the underlying cause of the c-Kit expression and signaling imbalance.

Synthetic cannabinoid WIN 55,212–2 inhibits growth and induces cell death of oral and pancreatic stem-like/poorly differentiated tumor cells

We report here that synthetic cannabinoid WIN 55,212–2 inhibits tumor cell proliferation and induces cell death of oral and pancreatic tumor cells, and the effect is much more pronounced on stem-like/poorly differentiated OSCSCs and MP2 cells when compared to well-differentiated OSCCs, and PL-12 tumor cells. In addition, WIN 55,212-2 decreases cell surface expression of CD44, CD54, MHC class I and PD-L1 on oral and pancreatic tumor cells with the exception of PD-L1 expression on well-differentiated PL-12 pancreatic tumor cells which exhibits an increase in the expression rather than a decrease. Overall, we demonstrate that WIN 55,212-2 has an increased targeting activity against cancer stem cells/poorly differentiated oral and pancreatic tumor cells when compared to well-differentiated tumor cells, and furthermore, such differences in function do not correlate with the levels of CB1 and CB2 receptor expression on tumor cells, suggesting it's function either through post-receptor mediated activation and/or yet-to-be identified novel receptors. Intraperitoneal (IP) delivery of WIN 55-212-2 in humanized BLT mice is found to impart an activating potential for NK cells demonstrating increased NK cell mediated cytotoxicity and secretion of IFN-γ in our preliminary experiments. These results not only suggest a direct targeting of CSCs/poorly differentiated tumors by WIN 55-212-2 but also by indirect targeting of such tumors through the activation and increased functions of NK cells.

Dynamic features of tapasin as revealed by structures of two tapasin/Fab complexes

Abstract Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst. Supported by the intramural research program of the NIAID, NIH

Auxilin is a novel susceptibility gene for congenital heart block which directly impacts fetal heart function

ObjectiveNeonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association...

Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2.

Melanocortin-3 receptor (MC3R) is a regulator of energy homeostasis, and interaction of MC3R and melanocortin-2 receptor accessory protein 2 (MRAP2) plays a critical role in MC3R signaling of mammals. However, the physiological roles of MC3R in teleosts are not well understood. In this study, qRT-PCR was used to measure gene expression. Radioligand binding assay was used to study the binding properties of topmouth culter MC3R (caMC3R). Intracellular cAMP generation was determined by RIA, and caMC3R expression was quantified with flow cytometry. We showed that culter mc3r had higher expression in the CNS. All agonists could bind and stimulate caMC3R to increase dose dependently intracellular cAMP accumulation. Compared to human MC3R, culter MC3R showed higher constitutive activity, higher efficacies, and Rmax to alpha-melanocyte-stimulating hormone (α-MSH), des-α-MSH, and adrenocorticotrophic hormone. Both caMRAP2a and caMRAP2b markedly decreased caMC3R basal cAMP production. However, only caMRAP2a significantly decreased cell surface expression, Bmax, and Rmax of caMC3R. Expression analysis suggested that MRAP2a and MRAP2b might be more important in regulating MC3R/MC4R signaling during larval period, and reduced mc3r, mc4r, and pomc expression might be primarily involved in modulation of MC3R/MC4R in adults. These data indicated that the cloned caMC3R was a functional receptor. MRAP2a and MRAP2b had different effects on expression and signaling of caMC3R. In addition, expression analysis suggested that MRAP2s, receptors, and hormones might play different roles in regulating culter development and growth.

Analyses of the interactions of tapasin and ERp57-tapasin proteins with PaSTa 1 and PaSTa 2 antibodies and MHC-I molecules

Abstract The intracellular loading of major histocompatibility complex class I (MHC-I) molecules with high affinity peptides is a pre-requisite for their cell surface display as ligands for T and NK cells. Peptide acquisition occurs in the ER within the peptide loading complex (PLC) and is accomplished through the concerted action of several proteins, most notably tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. In order to gain a mechanistic understanding of tapasin function as well as its apparent allelomorph specificity we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSTa 1 and PaSTa 2, and investigated their binding to human recombinant soluble tapasin and ERp57/tapasin by SPR methods. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. Based on these results we developed an MHC-I binding assay employing PaSTa 1-captured tapasin or ERp57/tapasin which reveals affinities in the micromolar range for selected MHC-I alleles. These affinities are notably weaker than those obtained for the structurally related but PLC-independent chaperone, TAPBPR (TAP-binding protein, related), and may reflect distinct evolutionarily derived functions for the two related molecules.

CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.

Abstract P803: Pituitary Adenylate Cyclase-Activating Polypeptide via Cyclic-AMP Inhibits Nogo-A by Internalizing Its Receptor NgR1

Introduction: After a stroke, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. They bind to the Nogo-A receptor 1 (NgR1) and induce the collapse of growth cones and inhibit neurite outgrowth. Since NgR1 is the receptor for a variety of axonal growth inhibitors, it is a crucial target for the prevention of axonal growth inhibition. Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic activities and increases neuritogenesis and synaptic plasticity. It enhances functional recovery after stroke in various animal models. Methods: Neuroscreen-1 (NS-1) cells were selected for this study as they produce rapid and robust neurite outgrowth with NGF. Cell surface NgR1 was detected using the indirect immunofluorescence method. The internalization of NgR1 was quantitated using the biotinylation method and Western immunoblotting. Results: Using the indirect immunofluorescence method, we found that PACAP (PACAP-38) induced a rapid decrease in the cell surface expression of NgR1 in NS-1 cells. The biotinylation method revealed that PACAP induced the internalization of NgR1. This internalization of NgR1 was blocked by pretreatment of NS-1 cells with SQ 22536, an inhibitor for adenylate cyclase, suggesting that cAMP plays a crucial role in the internalization of NgR1. The protein kinase A (PKA)-specific inhibitor KT5720 did not block PACAP-induced NgR1 internalization, whereas the exchange protein directly activated by cAMP (Epac)-specific inhibitor ESI-09 blocked this internalization. Collectively, this data suggests that PACAP-induced NgR1 internalization is independent of PKA but is dependent on Epac. The PACAP-induced decrease in cell surface expression of NgR1 and its internalization desensitized NS-1 cells to Nogo-66-induced growth cone collapse and enhanced neuritogenesis. Conclusion: Cyclic-AMP and Epac are involved in the PACAP-induced desensitization of neuronal cells to Nogo-A and increase in neuritogenesis. Since PACAP crosses the blood-brain barrier, it may be a useful therapeutic agent to overcome axonal growth inhibitors and enhance functional recovery after stroke.

Identification and characterization of two novel melanocortin-3 receptor mutations in Chinese obese individuals

The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R), known as neural melanocortin receptors, have been implicated to be critical components of the hypothalamic leptin-melanocortin pathway and related to obesity pathogenesis. In contrast to extensive evidence from physiologic, biological, genetic studies demonstrating that MC4R is a critical regulator in obesity, whether MC3R mutation causes obesity is still controversial. In the present study, we screened for coding variants in the MC3R gene of 176 obese individuals (mean BMI 34.84 ± 0.19 kg/m2) and 170 lean controls (mean BMI 20.70 ± 0.08 kg/m2) to assess the prevalence of MC3R mutations in a Chinese cohort. Two novel mutations, A33D (c.C98 > A) and A259T (c.G775 > A), were identified in two subjects with morbid obesity, respectively. A259T was also identified in the carrier's sibling. In vitro functional studies showed that A33D was defective in the cAMP signaling pathway, whereas A259T MC3R had defective maximal binding and cAMP generation in response to NDP- and α-MSH, likely due to decreased cell surface expression. In addition, we showed that A33D and A259T were biased receptors and defect in constitutive activation of ERK1/2 signaling through MC3R might be a cause for morbid obesity. Our sequencing and co-segregation studies combined with comprehensive functional analysis demonstrated that A259T might be predisposing to obesity. Further investigations in larger cohorts will be needed in order to define this association and the specific phenotypic characteristics resulting from these mutations.

Myotonic dystrophy kinase-related CDC42-binding kinase α, a new transferrin receptor type 2-binding partner, is a regulator of erythropoiesis.

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.

Palmitoylation of the Bovine Foamy Virus Envelope Glycoprotein Is Required for Viral Replication

Membrane proteins of enveloped viruses have been reported to undergo palmitoylation, a post-translational modification often having a critical role in the function of these viral proteins and hence viral replication. In this study, we report that the foamy virus (FV) envelope (Env) glycoprotein is palmitoylated. Specifically, we found that bovine foamy virus (BFV) Env (BEnv) is palmitoylated at amino acid positions C58 and C59 by BDHHC3 and BDHHC20 in a DHHC motif-dependent manner. In addition, mutations C58S and C58/59S significantly decrease cell surface expression of BEnv, subviral particle (SVP) egress, and its membrane fusion activity, thus ultimately inhibiting BFV replication. The C59S mutation exerts a minor effect in this regard. Taken together, these data demonstrate that the function of BEnv in the context of BFV replication is under the regulation of palmitoylation.

Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A

Coibamide A is a potent cancer cell toxin and one of a select group of natural products that inhibit protein entry into the secretory pathway via a direct inhibition of the Sec61 protein translocon. Many Sec61 client proteins are clinically relevant drug targets once trafficked to their final destination in or outside the cell, however the use of Sec61 inhibitors to block early biosynthesis of specific proteins is at a pre-clinical stage. In the present study we evaluated the action of coibamide A against human epidermal growth factor receptor (HER, ErbB) proteins in representative breast and lung cancer cell types. HERs were selected for this study as they represent a family of Sec61 clients that is frequently dysregulated in human cancers, including coibamide-sensitive cell types. Although coibamide A inhibits biogenesis of a broad range of Sec61 substrate proteins in a presumed substrate-nonselective manner, endogenous HER3 (ErbB-3) and EGFR (ErbB-1) proteins were more sensitive to coibamide A, and the related Sec61 inhibitor apratoxin A, than HER2 (ErbB-2). Despite this rank order of sensitivity (HER3 > EGFR > HER2), Sec61-dependent inhibition by coibamide A was sufficient to decrease cell surface expression of HER2. We report that coibamide A- or apratoxin A-mediated block of HER3 entry into the secretory pathway is unlikely to be mediated by the HER3 signal peptide alone. HER3 (G11L/S15L), that is fully resistant to the highly substrate-selective cotransin analogue CT8, was more resistant than wild-type HER3 but only at low coibamide A (3 nM) concentrations; HER3 (G11L/S15L) expression was inhibited by higher concentrations of either natural product. Time- and concentration-dependent decreases in HER protein expression induced a commensurate reduction in AKT/MAPK signaling in breast and lung cancer cell types and loss in cell viability. Coibamide A potentiated the cytotoxic efficacy of small molecule kinase inhibitors lapatinib and erlotinib in breast and lung cancer cell types, respectively. These data indicate that natural product modulators of Sec61 function have value as chemical probes to interrogate HER/ErbB signaling in treatment-resistant human cancers.