Hans Georg Mannherz

Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species

When cultured hepatocytes were incubated in cell culture medium at 4°C for up to 30 h and then returned to 37°C, blebbing of the plasma membrane, cell detachment, chromatin condensation and margination, enhanced nuclear stainability with Hoechst 33342, ruffling of the nuclear membrane, and DNA fragmentation occurred. Similar to hepatocytes, cultured liver endothelial cells exhibited blebbing, chromatin condensation and margination, marked nuclear condensation, and increased stainability with Hoechst 33342 when exposed to hypothermia/rewarming. In both cell types, the occurrence and extent of these alterations were dependent on the duration of the cold incubation period. This cold‐induced apoptosis was inhibited by hypoxia, by an array of free radical scavengers/antioxidants, and by iron chelators. However, the extent of the protection by the different antioxidants was different in the two cell types: iron chelators provided complete protection in liver endothelial cells but only partial protection in hepatocytes, whereas lipophilic antioxidants such as α‐tocopherol provided complete protection in both cell types. During cold incubation, and especially during rewarming, lipid peroxidation occurred. These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold‐induced apoptosis, with ROS formation being completely iron‐mediated in liver endothelial cells and partially iron‐mediated in hepatocytes.—Rauen, U., Polzar, B., Stephan, H., Mannherz, H. G., de Groot, H. Cold‐induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB J. 13, 155–168 (1999)

Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering

Tripartite Toxin Luminescent bacterial symbionts of nematode worms that attack insects have long stirred interest in their possibilities for biological control. The bacteria produce a family of toxins composed of at least three subunits that resemble a widely occurring class of bacterial toxins also produced by human pathogens. Lang et al. (p. 1139) have elucidated the mode of action and structural interactions of some of these tripartite protein toxins and found that they poison the cells actin cytoskeleton by catalyzing unusual reactions. One toxin mediated adenosine diphosphate (ADP)–ribosylation at threonine-148 to cause actin polymerization, another ADP-ribosylated Rho protein at glutamine-63, and both synergized to cause actin clustering and cell paralysis. A bacterial toxin targets and modifies the actin cytoskeleton in insect larvae. The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)–ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins.

A Specific 1:1 G-actin: DNAase I complex formed by the action of DNAase I on F-actin

In 1967 Lindberg [1] reported the isolation of a protein from calf spleen which forms a 1 : 1 complex with DNAase I thereby inhibiting its activity. Quite recently Lazarides and Lindberg [2] have shown that G-actin inhibits DNAase I specifically and that this inhibition may be a natural role of actin. Furthermore, Lazarides and Lindberg [2] demonstrated that this DNAase inhibitor and G-actin share a number physicochemical properties, e.g. comigration on polyacrylamide gels in the presence of SDS, similar amino acid composition and tryptic peptide maps, and the ability to form high molecular weight polymers. It was therefore thought to be of interest to examine whether DNAase I would effect the polymerization of G-actin and conversely whether polymerized actin (F-actin) would interact with DNAase I, since actin is found in the filamentous form in situations involving directed motion. Furthermore, it is important to examine the reversibility of the DNAase I -ac t in interaction.

Actin as target for modification by bacterial protein toxins.

Various bacterial protein toxins and effectors target the actin cytoskeleton. At least three groups of toxins/effectors can be identified, which directly modify actin molecules. One group of toxins/effectors causes ADP‐ribosylation of actin at arginine‐177, thereby inhibiting actin polymerization. Members of this group are numerous binary actin–ADP‐ribosylating exotoxins (e.g. Clostridium botulinum C2 toxin) as well as several bacterial ADP‐ribosyltransferases (e.g. Salmonella enterica SpvB) which are not binary in structure. The second group includes toxins that modify actin to promote actin polymerization and the formation of actin aggregates. To this group belongs a toxin from the Photorhabdus luminescens Tc toxin complex that ADP‐ribosylates actin at threonine‐148. A third group of bacterial toxins/effectors (e.g. Vibrio cholerae multifunctional, autoprocessing RTX toxin) catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments. Novel findings about members of these toxin groups are discussed in detail.

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases.

It was previously shown ( Eur J Cell Biol 69, 105–119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47–54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.

Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution

The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X‐ray crystallographic methods. The three‐dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy‐atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta‐protein with a beta‐pleated sheet in each domain. These sheets are surrounded by several alpha‐helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44‐46, 51, 52, 60‐62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.

FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers

Summary Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1–FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

Crystals of skeletal muscle actin: pancreatic DNAase I complex.

Skeletal muscle actin and the bovine pancreatic DNAase I inhibitor from calf-spleen are very similar by various criteria [ 1,2] . These include molecular weights, amino acid composition, tryptic fingerprints, and inhibition of DNAase I activity. DNAase I reacts with both G-actin, to form a stable (Ka ‘v 10’ M-l) 1: 1 complex, and polymeric F-actin producing depolymerisation with concomitant formation of the 1: 1 G-actin: DNAase I complex [3,4]. Crystals of the calf-spleen DNAase I inhibitor (now termed cytoplasmic actin) complexed with a 15 000 dalton protein (called profilin) suitable for an X-ray diffraction study have recently been described [2] . We describe below the crystallisation of the 1: 1 complex formed by rabbit skeletal muscle actin and bovine pancreatic DNAase I from polyethylene glycol solutions. Although the physiological significance of the interaction between skeletal muscle actin and DNAase I is not yet known, the elucidation of the structure of the complex formed by them is of particular importance. Firstly, actin plays a major role in muscular contraction and this complex provides a route to its three-dimensional structure. Secondly, DNAase I is one of the few proteins interacting with DNA that is well described [5] and only one other similar protein has been crystallised [6] .

Actin: from structural plasticity to functional diversity.

This article addresses the multiple activities of actin. Starting out with the history of actins discovery, purification and structure, it emphasizes the close relation between structure and function. In this context, we also point to unconventional actin conformations. Their existence in living cells is not yet well documented, however, they seem to play a special role in the supramolecular patterning that underlies some of the physiological functions of actin. Conceivably, such conformations may contribute to actins diverse activities in the nucleus that are poorly understood so far.