Mark A. L. Atkinson

Phosphorylation of the MAP Kinase ERK2 Promotes Its Homodimerization and Nuclear Translocation

The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts

The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in approximately sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in approximately 22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.

Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilins deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilins C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.

Correlation Between Self-Association Modes and GTPase Activation of Dynamin

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 × 1017 M−3. Stopped-flow fluorescence experiments show that the association rate constant for 2′(3′)-O-N-methylanthraniloyl (mant)GTP is 7.0 × 10−5 M−1 s−1 and the dissociation rate constant is 2.1 s−1, whereas the dissociation rate constant for mantdeoxyGDP is 93 s−1. We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

Measurements of asbestos burden in tissues.

Abstract:  Asbestos inhaled into the lung is recognized as a potential causal agent for the development of diseases in man. The diseases induced by asbestos include lung cancer, fibrosis of the lung (asbestosis), and extrapulmonary tumors including mesothelioma (a tumor of the serosal membrane), as well as fibrosis and other changes in the pleura linings. The cause of these diseases can often be more specifically linked to asbestos exposure once tissue burden of asbestos is established. The asbestos burden in tissue can be defined as the number of asbestos bodies and/or the numbers and types of asbestos fibers found in the tissue. In either of these cases the quality of information is directly dependent on the preparative techniques and instrumentation used in the analysis. The present article will discuss the significance of findings of tissue burden based on both these variables.

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(3–25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40°C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB4. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40°C.In addition, IL-8(3–25) was also able to decrease the binding of prehead IL-8 to neutrophils. IL-8(3–25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(3–25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.

Stability of ferruginous bodies in human lung tissue following death, embalmment, and burial.

The identification of asbestos bodies in tissue sections is an indicator of past exposure to longer asbestos fibers. These structures are formed in lung tissue as a consequence of interactions with pulmonary macrophages resulting in the deposition of a ferroprotein (ferruginous) coating on the fiber. While the process of ferruginous body formation is known to take months in animal tissue, there is no published information on the stability of ferruginous bodies in tissue following death. The material assessed in the present study was obtained from lung material collected from an exhumed body approximately 8½ mo after death, embalmment, and burial. Tissue sections were reviewed for the presence of asbestos bodies. Additional pieces of lung tissue were digested, with the digestate being evaluated by light microscopy for ferruginous bodies and by electron microscopy for uncoated asbestos fibers and core analysis of asbestos bodies. Classical ferruginous (asbestos) bodies were found in abundance in the tissue sections including in areas with fibrosis. The levels of uncoated asbestos fibers and classical appearing ferruginous bodies (asbestos bodies) were consistent with occupational levels of tissue burden. The data from this study indicate that ferruginous bodies remain morphologically stable within the tissue for months following death, embalmment, and burial. Thus the lung tissue from this exhumed individual was usable not only to pathologically confirm asbestosis but also to provide quantitative data of occupational exposure to asbestos.

Enzyme Kinetics and Characterization of Mouse Pancreatic Elastase

In the present study we have purified and characterized murine pancreatic elastase. The enzyme was extracted from acetone powders of mouse pancreas, fractionally precipitated with ammonium sulfate, and further purified by ion exchange chromatography to a single band on SDS-PAGE. The mouse enzyme exists in a proform, which was activated by removing a signal peptide by tryptic cleavage. The active form of mouse pancreatic elastase was shown by ultracentrifugation to have a molecular weight of 25.9 kDa and a frictional ratio of 1.26. The pH optimum for proteolytic activity was 8.0. Kinetic measurements were made with a variety of substrates and inhibitors and compared with elastases from other sources. The enzymatic properties and kinetic profiles for mouse pancreatic elastase were similar to other known serine elastases.

Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes.

We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.