Toxicity and metabolism of the chloral-derived mammalian alkaloid 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) in PC12 cells

Chloral-derived beta-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson's disease. The cytotoxicity of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED50 value of 230 microM. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-beta-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the beta-carboline ring 'plane' due to the high steric demand of the huge dichloromethyl group at C(1).     

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

A database search of the Paramecium genome reveals 34 genes related to Ca²⁺-release channels of the inositol-1,4,5-trisphosphate (IP₃) or ryanodine receptor type (IP₃R, RyR). Phylogenetic analyses show that these Ca²⁺ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP₃Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP₃Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca²⁺ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP₃ channel, a group II member (P. tetraurelia IP₃R_N-1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP₃R_N affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca²⁺ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca²⁺ as signaling molecule.Ca²⁺ is an important component of cell activity in all organisms, from protozoa to mammals. Thereby Ca²⁺ may originate from the outside medium and/or from internal stores (7, 18). Ca²⁺ release from internal stores is mediated by various Ca²⁺ release channels (CRCs), of which the inositol-1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) families have been studied most extensively (8, 9, 29, 63). IP₃Rs and RyRs have been identified in various metazoan organisms (reviewed in references 9, 28, and 104). According to these reviews, there exist three genetically distinct isoforms of each receptor type in mammals and orthologues have been identified in various nonmammalian vertebrates, e.g., frogs, chickens, and fish. RyRs and IP₃Rs were also cloned and sequenced in the invertebrates Drosophila melanogaster and Caenorhabditis elegans, which possess one copy of each receptor type.Functional evidence for Ca²⁺ release in response to ryanodine or IP₃ receptor agonists has been described in several unicellular systems. Treatment of permeabilized Plasmodium chabaudi parasites with IP₃ results in Ca²⁺ release, which is inhibited by the IP₃ receptor antagonist heparin (69). Another apicomplexan parasite, Toxoplasma gondii, responds to agonists and antagonists of both, ryanodine and IP₃ receptors, by mediating increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (56). Stimulation of Trypanosoma cruzi with carbachol results in increased [Ca²⁺]_i and IP₃ (59). IP₃ and cyclic ADP-ribose induces Ca²⁺ release in Euglena gracilis microsome fractions in a dose-dependent manner (61). In the giant algae Chara corallina and Nitrella translucens, IP₃ produces action potentials involving increased [Ca²⁺]_i (93). Treatment of vacuolar membrane vesicles from Candida albicans with IP₃ results in Ca²⁺ release, blocked by heparin and ruthenium red (14). IP₃ generates and maintains a Ca²⁺ gradient in the hyphal tip of Neurospora crassa and the IP₃-sensitive channels have been reconstituted and characterized with the planar bilayer method (87). In summary, these publications suggest that IP₃-dependent signaling pathways are conserved among unicellular organisms, including protozoa.Despite these data, the molecular characterization of IP₃ or ryanodine receptors in low eukaryotes is currently a challenge since the identification of orthologues has not been possible thus far, probably because of evolutionary sequence divergence (66). Traynor et al. (96) identified an IP₃ receptor-like protein, IplA, in Dictyostelium discoideum, which possesses regions related to IP₃R sequences, but thus far no evidence for IP₃ interaction exists. We have recently described an IP₃R in the ciliated protozoa Paramecium tetraurelia (referred to here as P. tetraurelia IP₃R_N) (53), with features characteristic of mammalian IP₃Rs in terms of topology and ability for IP₃ binding. The expression level of P. tetraurelia IP₃R_N is modulated by extracellular Ca²⁺ concentrations ([Ca²⁺]_o) and immunofluorescence studies reveal an unexpected localization to the contractile vacuole complex (CVC), the major organelle involved in osmoregulation (2). The ionic composition of the contractile vacuole fluid by ion-selective microelectrodes (91) suggests that the organelle plays a major role in expelling an excess of cytosolic Ca²⁺. Therefore, these IP₃Rs may here mediate a latent, graded reflux of Ca²⁺ for fine-tuning of [Ca²⁺]_i and thus serve [Ca²⁺] homeostasis (53).Besides [Ca²⁺] homeostasis, the Paramecium cell has to regulate a variety of well-characterized processes (75). This includes exocytosis of dense-core secretory vesicles (trichocysts) (71, 74, 99). Each cell possesses up to 1,000 trichocysts attached to the cell membrane. Their contents can be extruded synchronously in response to natural stimuli, i.e., predators (34, as confirmed by Knoll et al. [49]), to artificial polyamine secretagogues such as aminoethyldextran (AED) (78), to caffeine (48) or to the ryanodine substitute, 4-chloro-meta-cresol (4-CmC) (46). Their expulsion strictly depends on Ca²⁺ (10) and is accompanied by an increase of intracellular [Ca²⁺]_i (24, 47). This Ca²⁺ signal originates from rapid mobilization of cortical stores, the alveolar sacs (33, 64, 74), superimposed by Ca²⁺ influx (46, 72). It thus represents a SOC-type mechanism (SOC, store-operated Ca²⁺ entry) known from mammalian systems (81).Upon exocytosis stimulation ∼60% of their total Ca²⁺ is released from alveolar sacs (33). These are Ca²⁺ stores (90) represented by flat membrane compartments tightly attached at the cell membrane surrounding each trichocyst docking site. They possess a SERCA-type pump located at the membrane facing the cell center (36, 37) and a luminal high-capacity/low-affinity CaBP of the calsequestrin type (73). Thus far, Ca²⁺ release channels of these stores were identified only indirectly as cells respond by exocytosis to the RyR activators caffeine (54, 48) and 4-CmC (46). However, an involvement of conserved RyRs has remained questionable as ryanodine is not able to activate Ca²⁺ release from alveolar sacs, as is the case with IP₃ (54). Therefore, one of the most intriguing questions is the elucidation of the molecular nature of the channels mediating Ca²⁺ release from alveolar sacs upon stimulated exocytosis.In the present work we describe a novel family of CRCs (P. tetraurelia CRC-IV-1), whose members display several properties of the channels postulated above. In detail, the identified CRC-IV-1 channels localize to the alveolar sacs. Functional and fluorochrome analyses after gene silencing reveal that they are essential for mediating Ca²⁺ release and exocytosis in response to AED, caffeine, or 4-CmC. Their classification as “novel” CRC type is based on a restricted relationship to the C-terminal channel domains of IP₃Rs and RyRs. The overall size and the number of putative transmembrane domains resemble IP₃Rs, but N-terminal parts of CRC-IV-1 channels do not show any conservation, such as an IP₃-binding domain. Therefore, CRC-IV-1 channels represent distant relatives of IP₃Rs and RyRs and may belong to an ancestral Ca²⁺ signaling pathway.     

Calnexin, calreticulin, and ERp57

In eukaryotic cells, the endoplasmic reticulum (ER) plays an essential role in the synthesis and maturation of a variety of important secretory and membrane proteins. For glycoproteins, the ER possesses a dedicated maturation system, which assists folding and ensures the quality of final products before ER release. Essential components of this system include the lectin chaperones calnexin (CNX) and calreticulin (CRT) and their associated co-chaperone ERp57, a glycoprotein specific thiol-disulfide oxidoreductase. The significance of this system is underscored by the fact that CNX and CRT interact with practically all glycoproteins investigated to date, and by the debilitating phenotypes revealed in knockout mice deficient in either gene. Compared to other important chaperone systems, such as the Hsp70s, Hsp90s and GroEL/GroES, the principles whereby this system works at the molecular level are relatively poorly understood. However, recent structural and biochemical data have provided important new insights into this chaperone system and present a solid basis for further mechanistic studies.     

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) and related derivatives: chemistry and biochemical effects on catecholamine biosynthesis

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo, 2) is a mammalian alkaloid that readily originates in the human organism, by Pictet-Spengler condensation of endogenously present tryptamine (Ta) and the non-natural hypnotic agent trichloroacetaldehyde (chloral, Clo). Due to its structural analogy to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1), TaClo is discussed to possibly contribute to the pathogenesis of Parkinson's disease acting as an environmental toxin. Previous investigations on rats and neuronal cell cultures revealed 2 to be capable of inducing severe disturbances on the dopamine metabolism. In this paper, we report on the effects of 2 on the activity of tyrosine hydroxylase [L-tyrosine, tetrayhydropteridine/oxygen oxidoreductase (3-hydroxylating), EC 1.14,16.2; TH] in vitro using rat brain homogenates prepared from the TH-rich nucleus accumbens. TaClo (2) dose-dependently inhibited basal TH activity (IC(50)=3 microM), and after enzyme activation by pituitary adenylate cyclase-activating polypeptide (PACAP-27), it also reduced L-DOPA formation (IC(50)=15 microM). Moreover, two presumable TaClo metabolites, 2-methyl-TaClo (N-Me-TaClo, 3) and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2)-TH beta C, 4), which were synthesized in good yields, also proved to be potent inhibitors of TH, with the strongest effect on basal activity (similar to TaClo) being observed for 3 (IC(50)=3 microM). In contrast to TaClo, however, 3 and 4 showed biphasic effects after TH activation with PACAP-27, inducing a marked increase of enzyme activity in the nanomolar range (<0.1 microM), while TH activity was nearly completely blocked at high concentrations (IC(100)=0.1mM). An X-ray diffraction investigation on the 3-dimensional structure of the 1-CCl(2)-TH beta C-derived trifluoroacetamide 7 revealed the voluminous and quite rigid dichloromethylene substituent to be only moderately twisted out of the beta-carboline ring 'plane', thus resulting in an increased ring strain of the partially hydrogenated pyrido moiety accompanied by a strong steric hindrance of Cl(1), Cl(2), C(13), and N(2), which pushes the N-trifluoroacetyl group upwards to an even higher extent than for the TaClo-related trifluoroacetamide 8.     

Stimulation of platelet apoptosis by peptidoglycan from Staphylococcus aureus 113

Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30?min exposure to monomeric fraction (mPGN) (≥50?ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)β(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1?μM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.     

A heteromeric RNA-binding protein is involved in maintaining acrophase and period of the circadian clock

The RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits. One (named C1) contains three lysine homology motifs and the other (named C3) has three RNA recognition motifs. CHLAMY1 binds specifically to uridine-guanine-repeat sequences and its circadian-binding activity is controlled at the posttranslational level, presumably by time-dependent formation of protein complexes consisting of C1 and C3 or C1 alone. Here we have characterized the role of the two subunits within the circadian system by measurements of a circadian rhythm of phototaxis in strains where C1 or C3 are either up- or down-regulated. Further, we have measured the rhythm of nitrite reductase activity in strains with reduced levels of C1 or C3. In case of changes in the C3 level (both increases and decreases), the acrophase of the phototaxis rhythm and of the nitrite reductase rhythm (C3 decrease) was shifted by several hours from subjective day (maximum in wild-type cells) back towards the night. In contrast, both silencing and overexpression of C1 resulted in disturbed circadian rhythms and arrhythmicity. Interestingly, the expression of C1 is interconnected with that of C3. Our data suggest that CHLAMY1 is involved in the control of the phase angle and period of the circadian clock in C. reinhardtii.     

Hotspot-centric de novo design of protein binders

Protein-protein interactions play critical roles in biology, and computational design of interactions could be useful in a range of applications. We describe in detail a general approach to de novo design of protein interactions based on computed, energetically optimized interaction hotspots, which was recently used to produce high-affinity binders of influenza hemagglutinin. We present several alternative approaches to identify and build the key hotspot interactions within both core secondary structural elements and variable loop regions and evaluate the method's performance in natural-interface recapitulation. We show that the method generates binding surfaces that are more conformationally restricted than previous design methods, reducing opportunities for off-target interactions.     

The human tyrosine aminotransferase gene: characterization of restriction fragment length polymorphisms and haplotype analysis in a family with tyrosinemia type II

Summary Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5 to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3 end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.