Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

The human natural killer cell immune synapse

Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

N-methyl-d-aspartate receptor activation and visual activity induce elongation factor-2 phosphorylation in amphibian tecta: A role for N-methyl-d-aspartate receptors in controlling protein synthesis

N-methyl-d-aspartate receptor (NMDAR) activation has been implicated in forms of synaptic plasticity involving long-term changes in neuronal structure, function, or protein expression. Transcriptional alterations have been correlated with NMDAR-mediated synaptic plasticity, but the problem of rapidly targeting new proteins to particular synapses is unsolved. One potential solution is synapse-specific protein translation, which is suggested by dendritic localization of numerous transcripts and subsynaptic polyribosomes. We report here a mechanism by which NMDAR activation at synapses may control this protein synthetic machinery. In intact tadpole tecta, NMDAR activation leads to phosphorylation of a subset of proteins, one of which we now identify as the eukaryotic translation elongation factor 2 (eEF2). Phosphorylation of eEF2 halts protein synthesis and may prepare cells to translate a new set of mRNAs. We show that NMDAR activation-induced eEF2 phosphorylation is widespread in tadpole tecta. In contrast, in adult tecta, where synaptic plasticity is reduced, this phosphorylation is restricted to short dendritic regions that process binocular information. Biochemical and anatomical evidence shows that this NMDAR activation-induced eEF2 phosphorylation is localized to subsynaptic sites. Moreover, eEF2 phosphorylation is induced by visual stimulation, and NMDAR blockade before stimulation eliminates this effect. Thus, NMDAR activation, which is known to mediate synaptic changes in the developing frog, could produce local postsynaptic alterations in protein synthesis by inducing eEF2 phosphorylation.

Block of transmitter release by botulinum C1 action on syntaxin at the squid giant synapse

Electrophysiological, morphological, and biochemical approaches were combined to study the effect of the presynaptic injection of the light chain of botulinum toxin C1 into the squid giant synapse. Presynaptic injection was accompanied by synaptic block that occurred progressively as the toxin filled the presynaptic terminal. Neither the presynaptic action potential nor the Ca2+ currents in the presynaptic terminal were affected by the toxin. Biochemical analysis of syntaxin moiety in squid indicates that the light chain of botulinum toxin C1 lyses syntaxin in vitro, suggesting that this was the mechanism responsible for synaptic block. Ultrastructure of the injected synapses demonstrates an enormous increase in the number of presynaptic vesicles, suggesting that the release rather than the docking of vesicles is affected by biochemical lysing of the syntaxin molecule.

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type α1 subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α1D products, but only once an α1C product and never an α1S product. A full-length α1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α1D subunit was about 90% identical to those of the mammalian α1D subunits. In situ hybridization confirmed that the α1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α1D mRNA is 100–500 times more abundant than the α1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an α1D subunit. Furthermore, we propose that the α1D subunit plays a hitherto undocumented role at tonic synapses.

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

The Conserved Core of a Human SIR2 Homologue Functions in Yeast Silencing

Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2’s silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein’s core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast–human chimeras’ ability to function at only a subset of Sir2p’s target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.

Modulation of Life-span by Histone Deacetylase Genes in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Δ on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Δ sir3Δ resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Δ single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

SET1, A Yeast Member of the Trithorax Family, Functions in Transcriptional Silencing and Diverse Cellular Processes

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40–50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.

Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy

The activation of the silent endogenous progesterone receptor (PR) gene by 17-β-estradiol (E2) in cells stably transfected with estrogen receptor (ER) was used as a model system to study the mechanism of E2-induced transcription. The time course of E2-induced PR transcription rate was determined by nuclear run-on assays. No marked effect on specific PR gene transcription rates was detected at 0 and 1 h of E2 treatment. After 3 h of E2 treatment, the PR mRNA synthesis rate increased 2.0- ± 0.2-fold and continued to increase to 3.5- ± 0.4-fold by 24 h as compared with 0 h. The transcription rate increase was followed by PR mRNA accumulation. No PR mRNA was detectable at 0, 1, and 3 h of E2 treatment. PR mRNA accumulation was detected at 6 h of E2 treatment and continued to accumulate until 18 h, the longest time point examined. Interestingly, this slow and gradual transcription rate increase of the endogenous PR gene did not parallel binding of E2 to ER, which was maximized within 30 min. Furthermore, the E2–ER level was down-regulated to 15% at 3 h as compared with 30 min of E2 treatment and remained low at 24 h of E2 exposure. These paradoxical observations indicate that E2-induced transcription activation is more complicated than just an association of the occupied ER with the transcription machinery.

Ca2+/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-d-aspartate receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system

Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology.