The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.     

The role of the plasmalemmal dopamine and vesicular monoamine transporters in methamphetamine-induced dopaminergic deficits

Amphetamine (AMPH) and methamphetamine (METH) are members of a collection of phenethylamine psychostimulants that are commonly referred to collectively as "amphetamines." Amphetamines exert their effects, in part, by affecting neuronal dopamine transport. This review thus focuses on the effects of AMPH and METH on the plasmalemmal dopamine transporter and the vesicular monoamine transporter-2 in animal models with a particular emphasis on how these effects, which may vary for the different stereoisomers, contribute to persistent dopaminergic deficits.     

A rapid oxidation and persistent decrease in the vesicular monoamine transporter 2 after methamphetamine

Methamphetamine (METH) produces long-term decreases in markers of dopamine (DA) terminals in animals and humans. A decrease in the function of the vesicular monoamine transporter 2 (VMAT2) has been associated with damage to striatal DA terminals caused by METH; however, a possible mechanism for this decrease in VMAT2 function has not been defined. The current study showed that METH caused a rapid decrease to 68% of controls in VMAT2 protein immunoreactivity of the vesicular fraction from striatal synaptosomes within 1 h after a repeated high-dose administration regimen of METH. This decrease was associated with a 75% increase in nitrosylation of VMAT2 protein in the synaptosomal fraction as measured by nitrosocysteine immunoreactivity of VMAT2 protein. The rapid decreases in VMAT2 persisted when evaluated 7 days later and were illustrated by decreases in VMAT2 immunoreactivity and DA content of the vesicular fraction to 34% and 51% of control values, respectively. The decreases were blocked or attenuated by prior injections of the neuronal nitric oxide synthase inhibitor, S-methyl-l-thiocitrulline. These studies demonstrate that METH causes a rapid neuronal nitric oxide synthase-dependent oxidation of VMAT2 and long-term decreases in VMAT2 protein and function. The results also suggest that surviving DA terminals after METH exposure may have a compromised capacity to buffer cytosolic DA concentrations and DA-derived oxidative stress.     

Protein kinase A affects trafficking of the vesicular monoamine transporters in PC12 cells

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.     

Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.     

Synthesis and biological evaluation of 3-alkyl-dihydrotetrabenazine derivatives as vesicular monoamine transporter-2 (VMAT2) ligands

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [¹²⁵I]-iodovinyl-TBZ or [¹⁸F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 (K_i = 5.98 nM) and may be a useful lead compound for future structure-activity studies.     

分子伴侣的功能和应用

本文综述了分子伴侣的分类、功能、作用机理、研究现状及应用前景。分子伴侣是在生物大分子的折叠、组装、转运及降解等过程中起协助作用,参与协助抗原的呈递和遗传物质的复制、转录及构象的确立,但自身并不发生任何变化的一大类广泛存在于生物体内的蛋白质分子。随着对分子伴侣的进一步研究和相关知识的不断深入,分子伴侣在生物产品开发、物种改良、抗衰老,疾病预防、诊断和治疗以及环境监测方面具有广阔的前景。     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

The organic cation transporters (OCT1, OCT2, EMT) and the plasma membrane monoamine transporter (PMAT) show differential distribution and cyclic expression pattern in human endometrium and early pregnancy decidua

The non-neuronal monoamine transporters (OCT1, OCT2, EMT, and PMAT) play a key role in the clearance of monoamines from extracellular compartments. In a previous report we described endometrial distribution and cyclic variation of the vesicular monoamine transporter (VMAT2) mRNA and the neuronal norepinephrine transporter (NET) mRNA. In the present study we used in situ hybridization, real-time PCR and immunohistochemistry to reveal tissue distribution and cyclic variation of mRNA for the non-neuronal monoamine transporters in the human endometrium and early pregnancy decidua. We found that non-neuronal monoamine transporters are predominantly expressed in the stroma. The plasma membrane monoamine transporter (PMAT) mRNA expression peaked in the proliferative phase, whereas the extra-neuronal monoamine transporter (EMT) mRNA expression peaked in the secretory phase. The organic cation transporter 2 (OCT2) mRNA expression was exclusively detected in few scattered stromal cells and OCT1 mRNA was not detected at all. Our present results demonstrate that PMAT, EMT, and OCT2 transporters are expressed in the endometrial stroma and can potentially regulate reuptake of monoamines in general and histamine in particular. Taken together with our previous finding of VMAT2 mRNA in epithelial cells, we suggest a paracrine interaction between stromal and epithelial cells, which may modulate certain steps of the reproductive process.     

The induction mechanism of the molecular chaperone HSP70 in the gastric mucosa by Geranylgeranylacetone (HSP-inducer)

To elucidate the induction mechanism of HSP70 by geranylgeranylacetone (GGA), we investigated GGA specific binding proteins using a GGA-affinity column. Alteration of chaperone activity of HSP70 and binding affinity of HSP70 to heat shock factor-1 (HSF-1) was evaluated in the presence or absence of GGA. The binding domain of HSP70 to GGA was also analyzed. A 70-kDa protein eluted by 10 mM GGA from the GGA-affinity column was identical to constitutively expressed HSP70 on immunoblotting. GGA-binding domain of HSP70 was C-terminal of the protein as peptide-binding domain (HSP70C). The chaperone activity of HSP70 and recombinant HSP70C was suppressed by GGA. Furthermore, dissociation of the HSP70 from HSF-1 was observed in the presence of GGA. GGA preferentially binds to the C-terminal of HSP70 which binds to HSF-1. After dissociation of HSP70, free HSF-1 could acquire the ability to bind to HSE (the promoter region of HSP70) gene.     

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.     

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.     

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.     

Elevation of the Hsp70 chaperone does not effect toxicity in mouse models of familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) account for 10-20% of a familial form of amyotrophic lateral sclerosis (ALS). A common feature of SOD1 mutants is abnormal aggregation of the aberrant SOD1 in neurons and glia. We now report that in ALS transgenic mouse models the constitutively expressed heat shock protein 70 (Hsp70) is mislocalized into aggregates together with mutant SOD1 and ubiquitin. Forcing increased synthesis of Hsp70 ameliorates both aggregate formation and toxicity in primary motor neurons in culture. However, chronic increase in an inducible form of Hsp70 to about 10-fold its normal level is shown here not to affect disease course or pathology developed in mice from accumulation of any of three familial ALS causing SOD1 mutants with different underlying biochemical characteristics. Therefore, increasing Hsp70 to a level that is protective in mouse models of acute ischemic insult and selected neurodegenerative disorders is not sufficient to ameliorate mutant SOD1-mediated toxicity.     

GZ‐793A,a lobelane analog,interacts with the vesicular monoamine transporter‐2 to inhibit the effect of methamphetamine

(R)‐3‐[2,6‐cis‐Di(4‐methoxyphenethyl)piperidin‐1‐yl]propane‐1,2‐diol (GZ‐793A) inhibits methamphetamine‐evoked dopamine release from striatal slices and methamphetamine self‐administration in rats. GZ‐793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter‐2 (VMAT2). This study determined GZ‐793A's ability to evoke [³H]dopamine release and inhibit methamphetamine‐evoked [³H]dopamine release from isolated striatal synaptic vesicles. Results show GZ‐793A concentration‐dependent [³H]dopamine release; nonlinear regression revealed a two‐site model of interaction with VMAT2 (High‐ and Low‐EC₅₀ = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ‐793A‐evoked [³H]dopamine release, however, only at the High‐affinity site. Low concentrations of GZ‐793A that interact with the extravesicular dopamine uptake site and the High‐affinity intravesicular DA release site also inhibited methamphetamine‐evoked [³H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration‐response was evident with increasing concentrations of GZ‐793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ‐793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High‐affinity tetrabenazine‐ and reserpine‐sensitive site, dopamine release via a Low‐affinity tetrabenazine‐ and reserpine‐insensitive site, and a low‐affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ‐793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.

     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i?=?0.014–0.073?µM). Compound 15d exhibited the highest affinity (K_i?=?0.014?µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i?=?0.073?µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.     

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.