Protein Phosphorylation and APP Metabolism

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimer's amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimer's disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed A. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and A production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

Antisense suppression of a beta-galactosidase gene (TB G6) in tomato increases fruit cracking

Antisense suppression of a tomato beta-galactosidase gene (TBG6) was used to study its role in fruit development, cell wall-modification, and fruit firmness. TBG6 mRNA is highly abundant during the early stages of fruit development, but the levels decline sharply after the breaker stage with the start of the respiratory climacteric and a concomitant increase in ethylene production. Two antisense lines were obtained with significantly reduced levels of TBG6 mRNA at all stages of fruit development. At 30 d after pollination (dap), TBG6 mRNA levels were reduced by up to 98% and 88% in lines 6-2 and 6-10, respectively. Morphological phenotypes observed in the antisense lines included increased fruit cracking, reduced locular space, and a doubling in the thickness of the fruit cuticle. Two biochemical changes in antisense lines, compared with wild-type lines, were a reduction of exo-galactanase activity at the breaker +3 d stage and a reduction in the cell wall galactosyl content at the 20 dap stage. In addition, transgenic lines exhibited a 35-39% reduction in fruit firmness at the 20 dap stage, but their texture was equivalent to the wild type at 30 dap and beyond. Although the exact function of the TBG6 product is still unknown, these results implicate an important role for this enzyme in early fruit growth and development in tomato.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Modulation of 5-HT3 receptor-mediated response and trafficking by activation of protein kinase C

Modulation of neurotransmitter-gated membrane ion channels by protein kinase C (PKC) has been the subject of a number of studies. However, less is known about PKC modulation of the serotonin type 3 (5-HT3) receptor, a ligand-gated membrane ion channel that can mediate fast synaptic transmission in the central and peripheral nervous system. Here, we show that PKC potentiated 5-HT3 receptor-mediated current in Xenopus oocytes expressing 5-HT3A receptors and mouse N1E-115 neuroblastoma cells. In addition, using a specific antibody directed to the extracellular N-terminal domain of the 5-HT3A receptor, treatment with the PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA), significantly increased surface immunofluorescence. PKC also increased the amount of 5-HT3A receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3A receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. PMA potentiation is unlikely to occur via direct phosphorylation of the 5-HT3A receptor protein since the potentiation was not affected by point mutation of each of the putative sites for PKC phosphorylation. However, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibited PMA potentiation of 5-HT-activated responses in both N1E-115 cells and oocytes expressing 5-HT3A receptors. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhanced the PMA potentiation of 5-HT3A receptors. The observations suggest that PKC can modulate 5-HT3A receptor function and trafficking through an F-actin-dependent mechanism.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

Cellular and humoral responses to collagen-polyvinylpyrrolidone administered during short and long periods in humans

Collagen, particularly type I, and its related derivatives have been extensively employed in many areas of pharmacology. The present study was performed to determine the safety of collagen-polyvinylpyrrolidone (collagen-PVP) by in vitro and in vivo studies. Sera and peripheral blood cells from healthy donors without treatment and patients treated with collagen-PVP were evaluated. We observed that the biodrug does not stimulate lymphoproliferation or DNA damage in vitro, nor does it induce human anti-porcine type I collagen or anti-collagen-PVP antibodies in vivo. Furthermore, no hepatic or renal metabolic dysfunctions were observed when collagen-PVP was administered by intradermal or intramuscular routes in short- or long-term treatments. In conclusion, the present work shows that no cellular damage or immunological adverse effects (cellular and humoral) occurred during collagen-PVP treatment, even after more than 400 weeks of consecutive administrations.