Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Amphidoma languida (Amphidomatacea,Dinophyceae) with a novel azaspiracid toxin profile identified as the cause of molluscan contamination at the Atlantic coast of southern Spain

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC–MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160 μg AZA-1 eq. kg⁻¹ (reaching maximum levels of >500 μg AZA-1 eq. kg⁻¹ in Chamelea gallina and >250 μg AZA-1 eq. kg⁻¹ in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins.     

Binding of the Neuronal Protein B-50, but Not the Metabolite B-60, to Calmodulin

Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.     

Expression of a neuromodulin-β-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones

Cultured embryonic neurons share a number of characteristic morphological and physiological properties with their counterparts in vivo. For example, differentiating hippocampal neurons in culture develop two distinct classes of processes that serve as dendrites and axons. It has also been shown that the microtubule organization and composition in axons differs from those in dendrites, which may contribute to differential transport of macromolecules into axons or dendrites. We have expressed a neuromodulin--galactosidase fusion gene in cultured mesencephalic neurons in order to study the transport of the neurospecific protein neuromodulin into neurite growth cones. When -galactosidase alone was expressed in neurons, it was found in the cell bodies with diffuse neurite staining. In marked contrast, the neuromodulin--galactosidase fusion protein was rapidly transported into neurites and was concentrated in the growth cones. This system may provide a useful model for studying the structural domain(s) of neuromodulin that are required for transport and accumulation of neuromodulin in the growth cones of neurons.     

Various proteinase inhibitors decrease prolactin and growth hormone release by anterior pituitary cells

Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated.     

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis,Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

The role of gap junction membrane channels in development

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.