Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

Mechanisms of the anterograde trafficking of GPCRs: Regulation of AT1R transport by interacting proteins and motifs.

Anterograde cell surface transport of nascent G protein-coupled receptors (GPCRs) en route from the endoplasmic reticulum (ER) through the Golgi apparatus represents a crucial checkpoint to control the amount of the receptors at the functional destination and the strength of receptor activation-elicited cellular responses. However, as compared with extensively studied internalization and recycling processes, the molecular mechanisms of cell surface trafficking of GPCRs are relatively less defined. Here, we will review the current advances in understanding the ER-Golgi-cell surface transport of GPCRs and use angiotensin II type 1 receptor as a representative GPCR to discuss emerging roles of receptor-interacting proteins and specific motifs embedded within the receptors in controlling the forward traffic of GPCRs along the biosynthetic pathway.

Incidence and mortality of cervical cancer in China, 2013.

OBJECTIVE: Estimating the incidence and mortality rate of cervical cancer became necessary to establish prevention measures and healthy policies. The aim of this study was to estimate the updated incidence and mortality rate of cervical cancer in 2013 in China. METHODS: According to the evaluation criteria developed by the National Central Cancer Registry of China, the data submitted from 255 cancer registries met the required standards in 2013. Cervical cancer cases were retrieved from the national database and combined with the 2013 national population data. The age-standardized incidence and mortality rates were based on the demographic structure of the national census 2000 and Segi's world population. RESULTS: In 2013, the estimated number of new cases and deaths from cervical cancer were 100,700 and 26,400, respectively. The crude incidence of cervical cancer was 15.17/100,000. The age-standardized incidence rates based on the Chinese standard population (ASIRC) and the world standard population (ASIRW) were 11.30/100,000 and 10.30/100,000, respectively. The incidence of cervical cancer in urban areas was 15.62/100,000 and the ASIRC was 11.12/100,000. The incidence of cervical cancer in rural areas was 14.65/100,000 and the ASIRC was 11.47/100,000. The mortality rate of cervical cancer was 3.98/100,000. The age-standardized mortality rates based on the Chinese (ASMRC) and world standard populations (ASMRW) were 2.76/100,000 and 2.62/100,000, respectively. The mortality rate of cervical cancer in urban areas was 3.85/100,000 and in rural areas was 4.14/100,000. Cervical cancer incidence and mortality increased with age. Urban areas had a higher incidence of cervical cancer and lower mortality rates when compared with rural areas. CONCLUSIONS: Dynamic monitoring of cervical cancer incidence and mortality is the fundamental work of cervical cancer prevention and control. Cervical cancer is a serious issue in women's health, and prevention strategies need to be enhanced, such as human papilloma virus (HPV) vaccination and screening programs.

Vascular disease in mice with a dysfunctional circadian clock.

BACKGROUND: Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. A profound pattern exists in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Although the molecular components of circadian rhythms rhythmically oscillate in blood vessels, evidence of a direct function for the "circadian clock" in the progression to vascular disease is lacking. METHODS AND RESULTS: In the present study, we found increased pathological remodeling and vascular injury in mice with aberrant circadian rhythms, Bmal1-knockout and Clock mutant. In addition, naive aortas from Bmal1-knockout and Clock mutant mice exhibit endothelial dysfunction. Akt and subsequent nitric oxide signaling, a pathway critical to vascular function, was significantly attenuated in arteries from Bmal1-knockout mice. CONCLUSIONS: Our data reveal a new role for the circadian clock during chronic vascular responses that may be of significance in the progression of vascular disease.

A Naturally Occurring Splice Variant of GGA1 Inhibits the Anterograde Post-Golgi Traffic of α2B-Adrenergic Receptor.

The regulatory mechanisms of cell surface targeting of nascent G protein-coupled receptors (GPCRs) en route from the endoplasmic reticulum through the Golgi remain poorly understood. We have recently demonstrated that three Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins (GGAs) mediate the post-Golgi export of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and directly interact with the receptor. In particular, GGA1 interaction with α2B-AR is mediated via its hinge domain. Here we determined the role of a naturally occurring truncated form of GGA1 (GGA1t) which lacks the N-terminal portion of the hinge domain in α2B-AR trafficking and elucidated the underlying mechanisms. We demonstrated that both GGA1 and GGA1t were colocalized and mainly expressed at the Golgi. In marked contrast to GGA1, the expression of GGA1t significantly attenuated the cell surface export of newly synthesized α2B-AR from the Golgi and in parallel receptor-mediated signaling. Furthermore, we found that GGA1t formed homodimers and heterodimers with GGA1. More interestingly, GGA1t was unable to bind the cargo α2B-AR and to recruit clathrin onto the trans-Golgi network. These data provide evidence implicating that the truncated form of GGA1 behaviors as a dominant-negative regulator for the cell surface export of α2B-AR and this function of GGA1t is attributed to its abilities to dimerize with its wide type counterpart and to inhibit cargo interaction and clathrin recruitment to form specialized transport vesicles.

Specific TBC Domain-Containing Proteins Control the ER-Golgi-Plasma Membrane Trafficking of GPCRs.

G-protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins. However, the molecular mechanisms underlying their cell surface delivery after synthesis remain poorly understood. Here, we screen the TBC domain-containing proteins, putative Rab GTPase-activating proteins (GAPs), in the intracellular trafficking of GPCRs and identify several TBC proteins that activity-dependently regulate the anterograde transport, en route from the endoplasmic reticulum to the Golgi or from the Golgi to the cell surface, of several prototypic GPCR members without affecting other plasma membrane proteins. We also show that TBC1D6 functions as a GAP for Rab26, physically associates with Rab26, and attenuates Rab26 interaction with GPCRs. Furthermore, both overexpression and depletion of TBC1D6 inhibit the post-Golgi traffic of GPCRs. These data demonstrate important roles of the TBC proteins in forward trafficking of nascent GPCRs and reveal regulatory mechanisms of GPCR targeting to the functional destination.

Xiao-Shen-Formula, a Traditional Chinese Medicine, Improves Glomerular Hyper-Filtration in Diabetic Nephropathy via Inhibiting Arginase Activation and Heparanase Expression.

Hyperglycemia induces glomerular hyper-filtration, which contributes to the development of diabetic nephropathy (DN), a condition that remains a challenge for treatment. The present study investigated the effect of Xiao-Shen-Formula (XSF) used for treatment of renal injury in type 1 DN mice model induced by streptozotocin (STZ) and its underlying mechanism in cultured human glomerular endothelial cell (hGECs). Studies were performed using control, diabetic DN, DN treated with XSF groups (1 g/kg/d, LXSF or 3 g/kg/d, HXSF) for 6 weeks and hGECs were post-treated with mice serum containing HXSF (MS-HXSF) and arginase inhibitor (ABH, 100 µM) in high glucose medium. HXSF treatment restored STZ-induced renal hyper-filtration, glomerulosclerosis, renal microvascular remodeling and the increased levels of systemic reactive oxidative species and inflammatory cytokines, accompanied by preventing the decreased expression of glomerular heparin sulfate and the increased levels of cortical heparanase and argianse2 protein and arginase activity. In hGECs study, MS-HXSF ameliorated the enhancement in arginase activity, the protein/mRNA expression of heparanase, mRNA levels of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and permeability of hGECs monolayers as well as the depression of nitric oxide production. Besides all these protective effects, XSF blunted the mRNA expression of TNF-α in vivo and vitro studies as well, which was not changed by the post-treatment of ABH or HXSF plus ABH. This study demonstrated that the protective effect of XSF might be related with vascular prevention, anti-inflammation and anti-oxidation through intervening multi-targets including glomerular endothelial arginase-heparanase signaling pathway in DN model.

Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2.

The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export.

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

Regulation of α2B-Adrenerigc Receptor Export Trafficking by Specific Motifs.

Intracellular trafficking and precise targeting to specific locations of G protein-coupled receptors (GPCRs) control the physiological functions of the receptors. Compared to the extensive efforts dedicated to understanding the events involved in the endocytic and recycling pathways, the molecular mechanisms underlying the transport of the GPCR superfamily from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane are relatively less well defined. Over the past years, we have used α(2B)-adrenergic receptor (α(2B)-AR) as a model to define the factors that control GPCR export trafficking. In this chapter, we will review specific motifs identified to mediate the export of nascent α(2B)-AR from the ER and the Golgi and discuss the possible underlying mechanisms. As these motifs are highly conserved among GPCRs, they may provide common mechanisms for export trafficking of these receptors.

A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling.

The amphipathic helix 8 in the membrane-proximal C-terminus is a structurally conserved feature of class A seven transmembrane-spanning G protein-coupled receptors (GPCRs). Mutations of this helical motif often cause receptor misfolding, defective cell surface transport and dysfunction. Surprisingly, we demonstrated here that a single point mutation at Lys308 in helix 8 markedly enhanced the steady-state surface density of the angiotensin II type 1a receptor (AT1aR). Consistent with the enhanced cell surface expression, Lys308 mutation significantly augmented AT1aR-mediated mitogen-activated protein kinase ERK1/2 activation, inositol phosphate production, and vascular smooth muscle cell migration. This mutation also increased the overall expression of AT1aR without altering receptor degradation. More interestingly, Lys308 mutation abolished AT1aR interaction with ß-COP, a component of COPI transport vesicles, and impaired AT1aR responsiveness to the inhibition of Rab6 GTPase involved in the Golgi-to-ER retrograde pathway. Furthermore, these functions of Lys308 were largely dependent on its positively charged property. These data reveal previously unappreciated functions of helix 8 and novel mechanisms governing the cell surface transport and function of AT1aR.

Hepatic overexpression of the prodomain of furin lessens progression of atherosclerosis and reduces vascular remodeling in response to injury.

OBJECTIVE: Atherosclerosis is a complex disease, involving elevated LDL-c, lipid accumulation in the blood vessel wall, foam cell formation and vascular dysfunction. Lowering plasma LDL-c is the cornerstone of current management of cardiovascular disease. However, new approaches which reduce plasma LDL-c and lessen the pathological vascular remodeling occurring in the disease should also have therapeutic value. Previously, we found that overexpression of profurin, the 83-amino acid prodomain of the proprotein convertase furin, lowered plasma HDL levels in wild-type mice. The question that remained was whether it had effects on apolipoprotein B (ApoB)-containing lipoproteins. METHODS: Adenovirus mediated overexpression of hepatic profurin in Ldlr(-/-)mice and wild-type mice were used to evaluate effects of profurin on ApoB-containing lipoproteins, atherosclerosis and vascular remodeling. RESULTS: Hepatic profurin overexpression resulted in a significant reduction in atherosclerotic lesion development in Ldlr(-/-)mice and a robust reduction in plasma LDL-c. Metabolic studies revealed lower secretion of ApoB and triglycerides in VLDL particles. Mechanistic studies showed that in the presence of profurin, hepatic ApoB, mainly ApoB100, was degraded by proteasomes. There was no effect on ApoB mRNA expression. Importantly, short-term hepatic profurin overexpression did not result in hepatic lipid accumulation. Blood vessel wall thickening caused by either wire-induced femoral artery injury or common carotid artery ligation was reduced. Profurin expression inhibited proliferation and migration in vascular smooth muscle cells in vitro. CONCLUSION: These results indicate that a profurin-based therapy has the potential to treat atherosclerosis by improving metabolic lipid profiles and reducing both atherosclerotic lesion development and pathological vascular remodeling.

Circadian clock control of Nox4 and reactive oxygen species in the vasculature.

Recent studies have shown that circadian clock disruption is associated with pathological remodeling in the arterial structure and vascular stiffness. Moreover, chronic circadian disruption is associated with dysfunction in endothelial responses and signaling. Reactive oxygen species have emerged as key regulators in vascular pathology. Previously, we have demonstrated that circadian clock dysfunction exacerbates superoxide production through eNOS uncoupling. To date, the impact of circadian clock mutation on vascular NADPH oxidase expression and function is not known. The goal in the current study was to determine if the circadian clock controls vascular Nox4 expression and hydrogen peroxide formation in arteries, particularly in endothelial and vascular smooth muscle cells. In aorta, there was an increase in hydrogen peroxide and Nox4 expression in mice with a dysfunctional circadian rhythm (Bmal1-KO mice). In addition, the Nox4 gene promoter is activated by the core circadian transcription factors. Lastly, in synchronized cultured human endothelial cells, Nox4 gene expression exhibited rhythmic oscillations. These data reveal that the circadian clock plays an important role in the control of Nox4 and disruption of the clock leads to subsequent production of reaction oxygen species.

Ratio of miR-196s to HOXC8 messenger RNA correlates with breast cancer cell migration and metastasis.

Expression profiling has identified metastasis-associated microRNAs (miRNA) but technical limitations hinder the discovery of metastasis-suppressing miRNAs. In this study, we sought metastasis-suppressing miRNAs by functional screening. Individual miRNAs were lentivirally introduced into metastatic MDA-MB-231 breast cancer cells and analyzed for effects on cell migration, a critical step in cancer metastasis. Among 486 miRNAs screened, 14 were identified that included all of the members of the miRNA-196 family (miR-196a1, miR-196a2, and miR-196b). Enforced expression of miR-196a1/2 or miR-196b abrogated in vitro invasion and in vivo spontaneous metastasis of breast cancer cells, indicating that members of the miR-196 family are potent metastasis suppressors. We found that miR-196 inhibited the expression of transcription factor HOXC8. Functional linkage was implied by small interfering RNA-mediated knockdown of HOXC8, which suppressed cell migration and metastasis, and by ectopic expression of HOXC8, which prevented the effects of miR-196 on cell migration and metastasis. Unlike other metastasis-associated miRNAs that have been described, the expressions of miR-196 were not correlated with breast cancer cell migration or the metastatic status of clinical breast tumor specimens. Instead, we detected an excellent correlation between the ratio of miR-196 to HOXC8 messages and the migratory behavior of breast cancer cell lines as well as the metastatic status of clinical samples. Our findings identify miRNA-196s as potent metastasis suppressors and reveal that the ratio of miR-196s to HOXC8 mRNA might be an indicator of the metastatic capability of breast tumors.

Mutational analysis of highly conserved residues in the phage phiC31 integrase reveals key amino acids necessary for the DNA recombination.

BACKGROUND: Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombination of phiC31 integrase. METHODOLOGY/PRINCIPAL FINDINGS: To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage. CONCLUSIONS/SIGNIFICANCE: This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.

TAT-phiC31 integrase mediates DNA recombination in mammalian cells.

Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.