Corrine Seebart

Nap1: taking a closer look at a juggler protein of extraordinary skills

The nucleosome assembly protein Nap1 is used extensively in the chromatin field to reconstitute nucleosomal templates for structural and functional studies. Beyond its role in facilitating experimental investigation of nucleosomes, the highly conserved Nap1 is one of the best‐studied members of the histone chaperone group. Here we review its numerous functions, focusing mainly on its roles in assembly and disassembly of the nucleosome particle, and its interactions with chromatin remodeling factors. Its presumed role in transcription through chromatin is also reviewed in detail. An attempt is made to clearly discriminate between fact and fiction, and to formulate the unresolved questions that need further attention. It is beyond doubt that the numerous, seemingly unrelated functions of this juggler protein have to be precisely channeled, coordinated, and regulated. Why nature endowed this specific protein with so many functions may remain a mystery. We are aware of the enormous challenge to the scientific community that understanding the mechanisms underlying these activities presents.—Zlatanova, J., Seebart, C., Tomschik, M. Nap1: taking a closer look at a juggler protein of extraordinary skills. FASEB J. 21, 1294–1310 (2007)

Influence of ionizing radiation on crotoxin: Biochemical and immunological aspects

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35, 1193-1203, 1997.-Two sets of proteins have been purified from the venom of the Brazilian coral snake, Micrurus frontalis frontalis. One set has mol. wts, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the 8000-13,000 range and includes some proteins which are toxic to mice and others which are not. These proteins appear to be isoforms of postsynaptic toxins. The other set shows phospholipase A2 (PLA2) activity and the toxic members of this set promote hemorrhage in mice in a manner closely resembling that produced by PLA2s isolated from the venom of the Australian tiger snake (Notechis scutatus scutatus). These PLA2s migrate on SDS-PAGE with apparent mol. wts in the 18,000-22,000 range which is characteristic of PLA2s that have an alpha-helix D similar to pancreatic PLA2s. Elapid venom PLA2s of the type which typically migrate on SDS-PAGE with mol. wts in the 13,000-16,000 range and do not have alpha-helix D have not been detected in M. f. frontalis venom.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

A GYROXIN ANALOG FROM THE VENOM OF THE BUSHMASTER (LACHESIS MUTA MUTA)

Clinical observations of possible neurotoxic activity in bushmaster (Lachesis muta muta) envenomations, coupled with the accepted ancestral relationship of Lachesis to other crotalids, suggested that Lachesis venom might contain a crotoxin-like molecule. Crude venom and gel-filtration fractions showed modest reactivity in enzyme-linked immunosorbent assays using rabbit polyclonal antibodies raised against the basic subunit of crotoxin, but no reaction was detected with a murine monoclonal antibody raised against the same antigen. Phospholipase assays, LD50 determinations and SDS-polyacrylamide gel electrophoresis indicated the presence of non-toxic phospholipases, but no crotoxin homologs. A higher mol.wt, toxic protein (60,000) with an LD50 of 0.07 micrograms/g in mice was isolated and purified, which induced gyroxin-like, rapid rolling motions in mice. Its amino terminal sequence shows considerable amino acid sequence identity with gyroxin from the venom of Crotalus durissus terrificus and other serine proteases.

New toxins from the venom of the common tiger snake (Notechis scutatus scutatus)

Scutoxin A and B represent two isoforms of a new toxic protein from the venom of the Australian tiger snake, Notechis scutatus scutatus. Both isoforms, of apparent mol. wt 13,000, are less basic than either notexin or notechis II-5. They both have similar i.v. LD50-values in mice of ca 0.006 micrograms/g, and phospholipase activities of about 136 mumoles of fatty acid released/min/mg at 37 degrees C when acting on phosphatidylcholine in the presence of Triton X-100. Toxicities of the scutoxins are the same as notexin and about seven times more potent than notechis II-5. ELISAs and western blot analyses indicate that the new toxins are immunologically similar to notexin and notechis II-5, with phospholipase activities falling between these latter two proteins. When crude venom is initially passed over a gel filtration column, each scutoxin isoform co-elutes in a different fraction with notexin. Gel filtration experiments using purified samples of notexin and scutoxin have failed to demonstrate any evidence for the formation of higher mol. wt protein complexes. Peptide mapping suggests the presence of five glutamate residues in one of the protein isoforms. These findings, together with the high toxicity and active phospholipase levels, demonstrate that the new proteins are not the previously reported non-toxic and enzymatically inactive notechis II-1. The combination of gel filtration on Sephacryl S-200 and cation-exchange chromatography used to isolate the scutoxins also permits recovery of notexin and notechis II-5 in high purity.

Proteins isolated from the venom of the common tiger snake (Notechis scutatus scutatus) promote hypotension and hemorrhage

Notechis scutatus scutatus venom contains several toxic acidic proteins called HTa-i which promote hypotension and hemorrhage in mice. They have apparent mol. wts in the 18,000-21,000 range, i.v. LD50 values between 0.5 and 1.5 micrograms/g, and no detectable phospholipase, arginine esterase, proteolytic or hemolytic activities. A polyclonal antibody raised against HTg binds to other purified proteins, suggesting that they are isoforms of the same protein. Many other elapid crude venoms contain proteins which recognize the polyclonal antibody raised against HTg. Crotalid and viperid crude venoms do not recognize this antibody, although some of their component proteins are known to exhibit hypotensive and hemorrhagic activities. A combination of gel-filtration on Sephacryl S-200, cation-exchange and anion-exchange chromatography allows isolation of the N. s. scutatus proteins in high purity. They are the first hypotension-inducing proteins to be purified from an Australian elapid.

Spatially segregated transcription and translation in cells of the endomembrane-containing bacterium Gemmata obscuriglobus

Significance Eukaryotic (plant and animal) cells possess a nuclear membrane that separates the two stages of gene expression (transcription and translation), whereas prokaryotic (bacteria and archaea) cells lack the nuclear membrane barrier to colocated transcription and translation. However, cells of the bacterium Gemmata obscuriglobus possess extensive intracellular membranes, resulting in superficially eukaryote-like cellular complexity. We have found that a substantial amount of G. obscuriglobus translation is uncoupled from transcription, broadening our understanding of the spatial organization of bacterial gene expression, which currently is based entirely on a handful of model species. This broader understanding provides a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes. The dogma of coupled transcription and translation in bacteria has been challenged by recent reports of spatial segregation of these processes within the relatively simple cellular organization of the model organisms Escherichia coli and Bacillus subtilis. The bacterial species Gemmata obscuriglobus possesses an extensive endomembrane system. The membranes generate a very convoluted intracellular architecture in which some of the cell’s ribosomes appear to have less direct access to the cell’s nucleoid(s) than others. This observation prompted us to test the hypothesis that a substantial proportion of G. obscuriglobus translation may be spatially segregated from transcription. Using immunofluorescence and immunoelectron microscopy, we showed that translating ribosomes are localized throughout the cell, with a quantitatively greater proportion found in regions distal to nucleoid(s). Our results extend information about the phylogenetic and morphological diversity of bacteria in which the spatial organization of transcription and translation has been studied. These findings also suggest that endomembranes may provide an obstacle to colocated transcription and translation, a role for endomembranes that has not been reported previously for a prokaryotic organism. Our studies of G. obscuriglobus may provide a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes.

Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X

Abstract Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Venoms and morphology of western diamondback/mojave rattlesnake hybrids

Les hybrides F1 et F2 de C.a. et C.s. presentent des caracteristiques morphologiques et de composition chimique des venins semblables a celles des parents