SalI Sites Research Articles

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.

De novo IGF2 mutation on the paternal allele in a patient with Silver-Russell syndrome and ectrodactyly.

Although paternally expressed IGF2 is known to play a critical role in placental and body growth, only a single mutation has been found in IGF2. We identified, through whole-exome sequencing, a de novo IGF2 indel mutation leading to frameshift (NM_000612.5:c.110_117delinsAGGTAA, p.(Leu37Glnfs*31)) in a patient with Silver-Russell syndrome, ectrodactyly, undermasculinized genitalia, developmental delay, and placental hypoplasia. Furthermore, we demonstrated that the mutation resided on the paternal allele by sequencing the long PCR product harboring the mutation- and methylation-sensitive SmaI and SalI sites before and after SmaI/SalI digestion. The results, together with the previous findings in four cases from a single family with a paternally inherited IGF2 nonsense mutation and those in patients with variable H19 differentially methylated region epimutations leading to compromised IGF2 expression, suggest that the whole phenotype of this patient is explainable by the IGF2 mutation, and that phenotypic severity is primarily determined by the IGF2 expression level in target tissues.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

A simplified system for generating recombinant E3-deleted canine adenovirus-2

Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin–Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector.

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

TRANSFORMATION OF ACC SYNTHASE GENE IN TREE PEONY, PSACS1, INTO NICOTIANA TABACUM

Ethylene plays important roles in the regulation of a number of physiological and developmental processes, and is involved in flower opening and senescence of many species. Further researches have been demonstrating that 1-aminocyclopropane-1-carboxylate synthase (ACC synthase, ACS) is a key enzyme regulating the ethylene biosynthetic pathway. To clarify the function of PsACS1 in tree peony, we constructed the sense expression vector of PsACS1 and transferred it into Nicotiana tabacum. Total RNA from the petals of cut tree peony (Paeonia suffruticosa ‘Luoyang Hong’) was obtained using the CTAB method. The full length cDNA of PsACS1 was cloned by PCR with primers added BamHI and SalI site sequence and transformed into pGM-T vector. After digestion with BamHI and SalI, PsACS1 was ligated with plant expression vector pBI121N in the sense way and the sense plant expression vetor pBIN-35S::PsACS1 was successfully constructed. Moreover, pBIN-35S::PsACS1 was transferred into Agrobacterium tumefaciens GV3101 and then transformed into Nicotiana tabacum ‘NC89’ through leaf discs. The adventitious shoots and plants were obtained under the selection pressure of 100 mg/L kanamycin. PCR analysis confirmed that PsACS1 was successfully transferred into the genome of Nicotiana tabacum.

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

Down Syndrome Cell Adhesion Molecule (DSCAM) Associates with Uncoordinated-5C (UNC5C) in Netrin-1-mediated Growth Cone Collapse

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.

L-xylo-3-Hexulose Reductase Is the Missing Link in the Oxidoreductive Pathway for d-Galactose Catabolism in Filamentous Fungi

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

Alternative Splicing Regulates Kv3.1 Polarized Targeting to Adjust Maximal Spiking Frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

The Endoplasmic Reticulum (ER)-associated Degradation System Regulates Aggregation and Degradation of Mutant Neuroserpin

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.

Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.

Quantitative Analysis of Tat Peptide Binding to Import Carriers Reveals Unconventional Nuclear Transport Properties

A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin α (Impα) and importin β (Impβ) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impβ carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/β-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences.

Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

Disruption of the Chaperonin Containing TCP-1 Function Affects Protein Networks Essential for Rod Outer Segment Morphogenesis and Survival

Type II Chaperonin Containing TCP-1 (CCT, also known as TCP-1 Ring Complex, TRiC) is a multi-subunit molecular machine thought to assist in the folding of ∼ 10% of newly translated cytosolic proteins in eukaryotes. A number of proteins folded by CCT have been identified in yeast and cultured mammalian cells, however, the function of this chaperonin in vivo has never been addressed. Here we demonstrate that suppressing the CCT activity in mouse photoreceptors by transgenic expression of a dominant-negative mutant of the CCT cofactor, phosducin-like protein (PhLP), results in the malformation of the outer segment, a cellular compartment responsible for light detection, and triggers rapid retinal degeneration. Investigation of the underlying causes by quantitative proteomics identified distinct protein networks, encompassing ∼ 200 proteins, which were significantly affected by the chaperonin deficiency. Notably among those were several essential proteins crucially engaged in structural support and visual signaling of the outer segment such as peripherin 2, Rom1, rhodopsin, transducin, and PDE6. These data for the first time demonstrate that normal CCT function is ultimately required for the morphogenesis and survival of sensory neurons of the retina, and suggest the chaperonin CCT deficiency as a potential, yet unexplored, cause of neurodegenerative diseases.

Nucleocytoplasmic Shuttling of Dysbindin-1, a Schizophrenia-related Protein, Regulates Synapsin I Expression

Dysbindin-1 is a 50-kDa coiled-coil-containing protein encoded by the gene DTNBP1 (dystrobrevin-binding protein 1), a candidate genetic factor for schizophrenia. Genetic variations in this gene confer a susceptibility to schizophrenia through a decreased expression of dysbindin-1. It was reported that dysbindin-1 regulates the expression of presynaptic proteins and the release of neurotransmitters. However, the precise functions of dysbindin-1 are largely unknown. Here, we show that dysbindin-1 is a novel nucleocytoplasmic shuttling protein and translocated to the nucleus upon treatment with leptomycin B, an inhibitor of exportin-1/CRM1-mediated nuclear export. Dysbindin-1 harbors a functional nuclear export signal necessary for its nuclear export, and the nucleocytoplasmic shuttling of dysbindin-1 affects its regulation of synapsin I expression. In brains of sandy mice, a dysbindin-1-null strain that displays abnormal behaviors related to schizophrenia, the protein and mRNA levels of synapsin I are decreased. These findings demonstrate that the nucleocytoplasmic shuttling of dysbindin-1 regulates synapsin I expression and thus may be involved in the pathogenesis of schizophrenia.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.