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Hofmann, A., Osman, A., Leow, C.Y., Driguez, P., McManus, D.P., Jones, M.K. (2010)
BioEssays, in press
Abstract
In recent years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti-parasite therapeutics.
Annexins are ubiquitous proteins found in almost all organisms across all kingdoms. Here, we present an overview of novel annexins from parasitic organisms, and summarise their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a unique antigenic epitope for use as a vaccine. | | |
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Nolan, M.J., Hofmann, A., Jex, A.R., Gasser, R.B. (2010)
Mol. Cell. Probes, in press
Abstract
Predicting how point mutations in genes alter the tertiary and quarternary structure of proteins is central to a number of areas of molecular biology and has implications in relation to the function and evolution of molecules. In the present study, we theoretically assessed the effects of 20 point mutations detected previously in a region of the triose-phosphate isomerase gene (tpi) of the protozoan Giardia duodenalis on the three-dimensional structure of the ‘wild-type’ protein (TPI). Amino acid substitutions arising from codon variations were mainly located at surface-accessible sites or in hydrophobic pockets of TPI. None of the substitutions was predicted to exert a significant change to the fold or functionality of the enzyme, with the exception of one alteration (Arg100). Almost all substitutions were either conservative or semi-conservative, and retained or even improved the expected stability of the fold. Overall, the findings provide support for the “neutral theory”, which contends that evolution at the molecular level is not solely shaped by “Darwinian selection but also by random drift of selectively neutral or nearly neutral mutants”. | | | PubMed | DOI |
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Clemen, C.S, Tangavelou, K., Strucksberg, K.-H., Just, S., Gaertner, L., Regus-Leidig, H., Stumpf, M., Reimann, J., Coras, R., Morgan, R.O., Fernandez, M.-P., Hofmann, A., Müller, S., Schoser, B., Hanisch, F.-G., Rottbauer, W., Blümcke, I., von Hörsten, S., Eichinger, L., Schröder, R. (2010)
Brain, in press
Abstract
Mutations of the human VCP gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). We identified strumpellin as a novel VCP binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate
that strumpellin is a ubiquitously expressed dual compartment protein present in cytosolic and endoplasmic reticulum fractions. Over-expression or ablation of the wild-type strumpellin in wound healing assays caused significantly reduced wound closure velocities, whereas over-expression of the disease causing strumpellin
N471D mutant showed no functional effect. Strumpellin knock-down experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth.
Knock-down studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a pre-synaptic localization. We further identified strumpellin in
pathological protein aggregates in IBMPFD, various myofibrillar myopathies, and in cortical neurons of a Huntington disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that strumpellin along with VCP may have a concerted pathogenic role in various protein aggregate diseases. | | |
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Campbell, B.E., Rabelo, E.M., Hofmann, A., Hu, M., Gasser, R.B. (2010)
Mol. Cell. Probes 24, 178-189
Abstract
Full-length complementary DNA (cDNA) sequence (designated Hc-stp-1) encoding a serine/threonine phosphatase (Hc-STP-1) was isolated from Haemonchus contortus, a strongylid nematode parasite of small ruminants. Hc-stp-1 was shown to be transcribed in males of the adult and fourth larval stage, but not in females, early larval stages or eggs. The full-length gene (2854 bp) contained ten exons and nine introns, and encoded a cDNA of 951 bp. Comparisons of the conceptually translated protein (317 amino acids, estimated at ~35 kDa) with serine/threonine phosphatases (STPs) from other organisms revealed the presence of the conserved motif LRGNHE. Structural analysis, by comparative modelling, confirmed strict conservation of residues and features involved in catalytic activity, and variation in the ligand-binding interface. Phylogenetic analysis of amino acid sequence data revealed that Hc-STP-1 clustered with STPs from other nematodes (including Caenorhabditis elegans, Trichostrongylus vitrinus, Oesophagostomum dentatum, Ascaris suum and Brugia malayi) to the exclusion of STPs from other organisms. The protein was inferred to be most closely related to the PP1 class of STPs of C. elegans, within a group containing STPs encoded, amongst others, by the genes gsp-3 and gsp-4 in this free-living nematode. The functions of proteins GSP-3 and GSP-4 are known to be central to spermatogenesis and other male-specific processes in C. elegans. The findings from the present and previous studies support the proposal that Hc-stp-1 and its product play a significant role in reproductive and/or developmental processes in maturing or adult male H. contortus. | | | PubMed | DOI |
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Lopez, M., Paul, B., Hofmann, A., Morizzi, J., Wu, Q.K., Charman, S.A., Innocenti, A., Vullo, D., Supuran, C.T., Poulsen, S.-A. (2009)
J. Med. Chem. 52, 6421-6432
Abstract
In this paper we present a new class of carbonic anhydrase (CA) inhibitor that was designed to selectively target the extracellular domains of the cancer-relevant CA isozymes. The aromatic moiety of the classical zinc binding sulfonamide CA inhibitors is absent from these compounds and instead they incorporate a hydrophilic mono- or disaccharide fragment directly attached to the sulfonamide group to give S-glycosyl primary sulfonamides (1-10). The inhibition properties of these compounds at the physiologically abundant human CA isozymes I and II and cancer-associated IX and XII were determined and all compounds had moderate potency with Kis in the micromolar range. We present the crystal structures of anomeric sulfonamides 4, 7 and 10 and the sugar sulfamate drug topiramate in complex with human recombinant CA II. From these structures we have obtained valuable insights into ligand-protein interactions of these novel carbohydrate-based sulfonamides with CA. | | | PubMed | DOI |
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Georgousakis, M., Hofmann, A., McMillan, D., Batzloff, M., Sriprakash, S. (2009)
Vaccine 27, 6799-6806
Abstract
A conformationally restricted B-cell epitope has been identified from the major group A streptococcal virulence factor, the M protein, as a potential safe vaccine candidate. To maintain alpha-helical secondary structure, the minimal epitope is flanked with heterologous sequences to produce the chimeric vaccine candidate J14. As a strategy towards developing an affordable multivalent GAS vaccine, we have expressed J14 recombinantly with a second GAS protective antigen H12 (rJ14H12). When administered to mice sub-cutaneously, the fusion protein stimulated a strong serum IgG response to the H12 component, but J14 was poorly immunogenic. To increase the immunogenicity of J14 when expressed with the model fusion partner, amino acid modifications were made to the initial recombinant construct to produce rJJo. These changes stabilised the alpha-helical conformation of the recombinant antigen as assessed by circular dichroism. Mice immunised with rJJoH12, the fusion protein incorporating JJo, effectively stimulated a humoral response to both of the included antigens. This data supports the feasibility of developing a multivalent vaccine incorporating the conformationally restricted protective antigen J14. | | | PubMed | DOI |
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Braunewell, K.-H., Paul, B., Altarche-Xifro, W., Noack, C., Lange, K., Hofmann, A. (2010)
Aust. J. Chem. 63, 350-356
Abstract
The subcellular membrane localisation of neuronal calcium sensor (NCS) proteins in living cells, such as VILIP-1 and VILIP-3, differs substantially. We have followed the hypothesis that the differential localisation might be due to specific binding capabilities of individual VILIPs for phosphatidylinositol phosphates (PIPs). Several highly conserved lysine residues in the N-terminal region could provide favourable electrostatic interactions.
Molecular modelling results support a binding site for phospho-inositides in the N-terminal area of VILIP-1, and the involvement of the conserved N-terminal lysine residues in binding the phospho-inositol head group. Experimentally, the binding of VILIP-1 to inositol derivatives was tested by a PIP strip assay, which showed the requirement of phosphorylation of the inositol group for the interaction of the protein with PIPs. Monolayer adsorption measurements showed a preference of VILIP-1 binding to PI(4,5)P2 over PI(3,4,5)P3. The co-localisation of VILIP-1 with PI(4,5)P2 at the cell surface membrane in hippocampal neurons further supports the idea of direct interactions of VILIP-1 with PIPs in living cells. | | | DOI |
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Xavier, C.P., Rastetter, R.H., Stumpf, M., Rosentreter, A., Müller, R., Reimann, J., Cornfine, S., Linder, S., van Vliet, V., Hofmann, A., Morgan, R.O., Fernandez, M.P., Schröder, R., Noegel, A.A., Clemen, C.S. (2009)
J. Mol. Biol. 393, 287-299
Abstract
Coronin-1C (synonyms: coronin-3, CRN2), a WD40 repeat containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here we report on the identification and functional characterization of two novel coronin-1C isoforms, referred to as CRN2i2 and CRN2i3. These isoforms, which arise from an alternative first exon identified in intron 1 of the coronin-1C gene, associate with F-actin. Gel filtration assays suggest that the largest isoform 3 exists as a monomer,
whereas the conventional isoform 1 and isoform 2 both form trimers. Structural
modelling predicts that this difference of the oligomerization state results from an interaction of the elongated N-terminus of CRN2i3 with its C-terminal extension.
Analyses of the coronin-1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors. CRN2i3 is expressed in mature skeletal muscle tissue and in differentiated myogenic cell lines. In human skeletal muscle CRN2i3 is a structural component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. In addition, CRN2i1 and i2 are enriched within the F-actin core structure of podosomes. Our findings postulate a role of CRN2 isoforms in the structural and
functional organization of F-actin in highly ordered protein complexes.
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Cantacessi, C., Campbell, B.E., Visser, A., Geldhof, P., Nolan, M.J., Nisbet, A.J., Matthews, J.B., Loukas, A., Hofmann, A., Otranto, D., Sternberg, P.W., Gasser, R.B. (2009)
Biotechnol. Advances 27, 376-388
Abstract
A wide range of proteins belonging to the SCP/TAPS “family” has been described for various eukaryotic organisms, including plants and animals (vertebrates and invertebrates, such as helminths). Although SCP/TAPS proteins have been proposed to play key roles in a number of fundamental biological processes, such as host-pathogen interactions and defence mechanisms, there is a paucity of information on their genetic relationships, structures and functions and there is no standardised nomenclature for these proteins. A detailed analysis of the relationships of members of the SCP/TAPS family of proteins, based on key protein signatures, could provide a foundation for investigating these areas. In this article, we review the current state of knowledge of key SCP/TAPS proteins of eukaryotes, with an emphasis on those from parasitic helminths, undertake a comprehensive, systematic phylogenetic analysis of currently available full-length protein sequence data (considering characteristic protein signatures or motifs) to infer relationships and provide a framework (based on statistical support) for the naming of these proteins. This framework is intended to guide genomic and molecular biological explorations of key SCP/TAPS molecules associated with infectious diseases of plants and animals. In particular, fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new and innovative approaches for the control of parasitic diseases, with important biotechnological outcomes. | | | PubMed | DOI |
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Laohavisit, A., Mortimer, J.C., Demidchik, V., Coxon, K.M., Stancombe, M.A., Macpherson, N., Brownlee, C., Hofmann, A., Webb, A.A.R., Miedema, H., Battey, N.H., Davies, J.M. (2009)
Plant Cell 21, 479-493
Abstract
Regulation of reactive oxygen species (ROS) and cytosolic free calcium ([Ca2+]cyt) is central
to plant function. Annexins are small proteins capable of Ca2+-dependent membrane-binding
or insertion. They possess structural motifs that could support both peroxidase activity and
calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when
added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological
profile was consistent with annexin activation (at the extracellular plasma membrane face) of
Arabidopsis Ca2+-permeable non-selective cation channels. Secreted annexins could therefore
modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they
were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma
membrane. Here they generated an instantaneously-activating Ca2+-permeable conductance at
mildly acidic pH, sensitive to verapamil and Gd3+ with a Ca2+ to K+ permeability ratio of 0.36.
These results suggest that cytosolic annexins create a Ca2+ influx pathway directly,
particularly during stress responses involving acidosis. Maize annexin preparation also
demonstrated in vitro peroxidase activity which appeared independent of heme association. In
conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport
pathways, regulate [Ca2+]cyt and in vitro may function as peroxidases. | | | PubMed | DOI | Publisher |
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Wang, Y.-Q., Feechan, A., Yun, B.-W., Shafiei, M., Hofmann, A., Taylor, P., Xue, P., Yang, F.-Q., Xie, Z.-S., Pallas, J.A., Chu, C.-C., Loake, G. (2009)
J. Biol. Chem. 284, 2131-2137
Abstract
Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this RedOx-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid binding protein 3 (AtSABP3) at cysteine (C)280, suppressing both binding of the immune activator, salicylic acid (SA) and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance. | | | PubMed | DOI | PDF |
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Hofmann, A. (2009)
J. Appl. Crystallogr. 42, 137-139
Abstract
ACDP is a Java application for processing protein circular dichroism (CD) spectra either individually or within a series. Data processing includes spectrum subtraction ('baseline correction'), conversion of the raw CD into units of mean residue ellipticity, wavelength monitoring and graphical inspection. Three different algorithms for secondary structure deconvolution have been implemented, and spectra can be analysed without the need for data reformatting using a neural network approach, variable selection or linear combination of prototype spectra. The application is written entirely in Java and is thus portable to a wide variety of platforms, requiring only the Java Runtime Environment. | | | DOI | More |
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Hu, N.-J., Yusof, A. M., Winter, A., Osman, A., Reeve, A. K., Hofmann, A. (2008)
J. Biol. Chem. 283, 18314-18322
Abstract
Plant annexins show distinct differences when compared to their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins.
The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.5 Å resolution, and shows three metal ions coordinated in the first and fourth repeat in type II and III binding sites. While the protein has no detectable affinity for calcium in solution in the presence of phospholipid vesicles, we determined a stoichiometry of four calcium ions per protein molecule using isothermal titration calorimetry. Further analysis of the crystal structure shows that binding of a fourth calcium ion is structurally possible in the DE loop of the first repeat. Data from this study are in agreement with the canonical membrane-binding of annexins which is facilitated by the convex surface associating with the phospholipid bilayer by a calcium bridging mechanism. In annexin Gh1, this membrane-binding state is characterised by four calcium bridges in the I/IV module of the protein, and direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane.
Analysis of the protein fold stability reveals that the presence of calcium lowers the thermal stability of plant annexins. Furthermore, an additional unfolding step was detected at lower temperatures, which can be explained by the anchoring of the N-terminal domain to the C-terminal core by two conserved hydrogen bonds. | | | PubMed | DOI | PDF | Publisher |
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Hu, N.-J., Bradshaw, J., Lauter, H., Buckingham, J., Solito, E., Hofmann, A. (2008)
Biophys. J. 94, 1773-1781
Abstract
Annexins constitute a family of calcium-dependent membrane binding proteins, and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation, and has been implicated in mediating the membrane aggregation behaviour of this protein. While the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane binding site of annexin A1.
The present study used circular diochroism spectroscopy to show that a rat annexin A1 N-terminal peptide possess random coil structure in aqueous buffer, but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveal an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane.
Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures, and that this conformation enables favourable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor. | | | PubMed | DOI | PDF | Publisher |
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McArdle, B., Hofmann, A. (2008)
The Coronin Family of Proteins , 56-71
Abstract
Until recently, structural information about coronins was scarce and the earlier identification of five WD40 repeats gave rise to a structural prediction of a five-bladed beta propeller for the N-terminal domain of these proteins. More detailed analyses revealed the presence of seven WD40 repeats and the hypothesis of a seven-bladed beta propeller structure. This model has recently been validated due to structural information from crystal structures of C-terminally truncated coronin 1, as well as its C-terminal coiled coil domain. Further structural information is available only indirectly from binding and functional studies.
Phosphorylation at distinct serine and tyrosine residues seems to be a common theme for various coronins. There are indications that this modification regulates the quaternary structure of coronin 3, and thus has implications for the cellular localisation and the general link between signalling and cytoskeletal remodelling. Similarly, phosphorylation-dependent sorting sequences recently discovered on coronin 7 might prove important for the molecular mechanisms of the longer coronins.
A matter that will require further clarification is the localisation of protein binding sites on coronins. While earlier reports presented a rather diverse map of actin binding sites, more recent studies, including the crystal structure of the coronin 1 N-terminal domain, deliver more detailed information in this respect. Interaction sites for other target proteins, such as Arp2/3, remain to be identified. Also, while membrane binding is a known feature of coronins, further details as to the binding sites and molecular level events remain to be elucidated. The N-terminal WD40 repeat domain seems to be the membrane-interacting domain, but other domains might provide regulatory effects, most likely by post-translational modification, in a fashion that is specific for each coronin.
In this chapter, we provide a structural overview of coronins 1, 2, 3 and 7, and also present results of our recent efforts to obtain structural models of coronins 3 and 7. Possible implications of these models on the function of these proteins are discussed. | | | PubMed | PDF | Publisher |
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Winter, A., Hofmann, A. (2008)
Curr. Chem. Biol. 2, 11-19
Abstract
Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that localise to different compartments of the cell. They have been implicated in important cellular processes such as cellular signalling and transcriptional control, apoptosis, cellular senescence, early development of Caenorhabditis elegans and mitochondrial biogenesis.
In yeast, mammals and C. elegans there exist at least two homologous prohibitin proteins (yeast: PHB1, PHB2; human: BAP32, BAP37), which assemble into high molecular weight complexes of about 1.2 MDa in the inner mitochondrial membrane. Experimentally determined structural information about these proteins has been elusive for a long time. Recently, however, the biogenesis and architecture of the yeast prohibitin complex has been analysed and yielded ring-shaped structures as visualised by single particle electron microscopy. Structural details at atomic level remain to be determined, but a first step into this direction is provided by modelling approaches. Prohibitins consist of three domains, an N-terminal transmembrane helix, a middle (PHB) domain and a C-terminal coiled coil domain. The PHB domain is the landmark feature within the super-family of SPFH (stomatin/prohibitin/flotillin/HflK/C) domain proteins. The recently determined NMR structure of mouse flotillin-2 provides a first access to structural details of prohibitins.
While the first functional role attributed to prohibitins was the regulation of cellular senescence, DNA transcription and tumour cell growth, there is recent evidence that they also can act as markers for adipose tissue. In a mouse model, an apoptotic peptide targeted at prohibitin was successful in reversing obesity. An extracellular complex containing both BAP32 and BAP37 was found to bind to the Vi capsular polysaccharide, first identified as a virulence antigen of Salmonella typhi, suggesting a key role for both proteins in infection with S. typhi. Furthermore, the interaction of prohibitin with compounds activating melanin production has placed these proteins at a central position in melanogenesis, and further implicates mitochondria in signalling pathways of the pigmentation process. Accumulating evidence suggests that prohibitins are implicated in mitochondrial, age and oxidative stress-related diseases, as well as in immunity and inflammation, cancer and cancer-like diseases, obesity, and drug resistance.
The complementary interplay between structural and chemical biology will provide important insights into the molecular mechanisms of prohibitins and, more generally, the functions of mitochondria in living cells. This review discusses the current state of knowledge about prohibitins, and provides a vision for further developments in the field of these eminently important proteins.
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Rosentreter, A., Hofmann, A., Xavier, C.-P., Stumpf, M., Noegel, A.A., Clemen, C.S. (2007)
Exp. Cell Res. 313, 878-895
Abstract
The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3 mediated events. | | | PubMed | DOI | PDF |
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Winter, A., Kämäräinen, O., Hofmann, A. (2007)
Proteins 68, 353-362
Abstract
Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that mainly localize to the mitochondria. They have been implicated in important cellular processes such as cellular signalling and transcriptional control, apoptosis, cellular senescence, and mitochondrial biogenesis.
Using molecular modelling techniques, we have generated structural models of human prohibitins BAP32 and BAP37 which have previously been shown to exist as large ring-like oligomers in the membrane-bound state. The middle domain of prohibitins is evolutionary conserved in the family of SPFH (PHB) domain proteins. Based on the known structure of flotillin-2, another member of the SPFH domain family, we have generated homology models for BAP32 and BAP37, and elucidated the implications for formation of high molecular weight oligomers. A model for the dimeric building block of BAP32:BAP37 for such assemblies was generated and its stability scrutinized by molecular dynamics simulations. The model of BAP32 was also analyzed as to potential ligand binding sites and the previously identified ligand melanogenin was docked into a membrane-proximal cavity.
The results are discussed in the context of prohibitin interactions with mitochondrial AAA-proteases and we suggest two possible interaction interfaces between the BAP32:BAP37 building block and the protease.
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Hübbers, C.U., Clemen, C.S., Kesper, K., Böddrich, A., Hofmann, A., Kämäräinen, O., Tolksdorf, K., Stumpf, M., Reichelt, J., Roth, U., Krause, S., Watts, G., Kimonis, V., Wattjes, M.P., Reimann, J., Thal, D.R., Biermann, K., Evert, B.O., Lochmüller, H., Wanker, E.E., Schoser, B.G.H., Noegel, A.A., Schröder, R. (2007)
Brain 130, 381-393
Abstract
Mutations in the valosin containing protein (VCP, p97) gene on chromosome 9p13-p12 cause
a late-onset form of autosomal dominant inclusion body myopathy associated with Paget
disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological
consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human
striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes
and filamentous VCP- and ubiquitin-positive cytoplasmic and nuclear protein aggregates.
Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of
dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle
cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not
detected in primary IBMPFD myoblasts or in transient and stable transfected cells using
wildtype-VCP and R93C-, R155 H-, R155C-VCP mutants. Glutathione S-transferase (GST)
pull-down experiments showed that all three VCP mutations do not affect the binding to
Ufd1, Npl4, and ataxin-3. Structural analysis demonstrated that R93 and R155 are both
surface-accessible residues located in the center of cavities that may enable ligand binding.
Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the
VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the
identification of cyclic sugar compounds with high binding scores. The latter findings provide
a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD. | | | PubMed | DOI | PDF |
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Yusof, A.M., Jaenicke, E., Pedersen, J.S., Noegel, A.A., Schleicher, M., Hofmann, A. (2006)
J. Mol. Biol. 362, 1072-1081
Abstract
Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin
filament dynamics and a variety of developmental and morphological processes. The protein exists as a high-molecular
weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for
crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of
oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal
helix-barrel domain are involved in inter-molecular interactions with the N- or C-terminal domains. Abolishing
these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the
protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with
mutations in the extreme N-terminal region of Dictyostelium CAP which did not show significantly altered
oligomerisation behaviour. Constructs with mutations in the earlier identified protein-protein interaction interface
on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins
indicates that the mutations did not affect their overall fold. Further studies point to the correlation between
stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable
protein. | | | PubMed | DOI | PDF |
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Winter, A., Yusof, A.M., Gao, E., Yan, H.L., Hofmann, A. (2006)
FEBS J. 273, 3238-3247
Abstract
Annexin B1 from Cysticercus cellulosae has recently been identified by immunological screening in an
attempt to find novel antigens for vaccine development against cysticercosis. The protein possesses anticoagulant
activity and carries a significant therapeutic potential due to its thrombus-targeting and thrombolytic properties.
We have investigated the biochemical properties of annexin B1 using liposome- and heparin-sepharose co-pelleting
assays, as well as CD spectroscopy. The calcium-dependent binding to acidic phospholipid membranes is reminiscent
of other mammalian annexins with a clear preference for high phosphatidylserine content. A unique property of
annexin B1 is its ability to bind to liposomes with high phosphatidylserine content in the absence of calcium which
might be due to the presence of several basic residues on the convex protein surface that harbours the membrane
binding loops. Annexin B1 demonstrates lectin properties and binds to heparin sepharose in a cooperative, calcium-
dependent manner. While this binding is reversible to a large extent, a small fraction of the protein remains bound
to the glycosaminoglycan even in the presence of high concentrations of EDTA. Analogous to annexin A5, we propose a
model of heparin wrapped around the protein thereby engaging in calcium-dependent and -independent interactions.
While the calcium-independent heparin binding sites identified in annexin A5 are not conserved, we hypothesise
three possible sites in annexin B1. Results from CD spectroscopy and thermal denaturation indicate that, in
solution, the protein binds calcium with a low affinity which leads to a slight increase in folding stability. | | | PubMed | DOI | PDF | Publisher |
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Brackmann, M., Hofmann, A., Braunewell, K.-H. (2006)
Neuronal calcium sensor proteins - Molecular Anatomy and Physiology of Proteins , 115-135
Abstract
The subfamily of VILIPs, with its members VILIP-1, -2, -3, hippocalcin and neurocalcin delta,
has been implicated in the modulation of a variety of intracellular signalling cascades. This
chapter will focus on two proteins, VILIP-1 and VILIP-3, which have recently been characterised
in more detail, and compare them to other members of the subfamily. Comparative studies have shown
that the proteins are predominantly expressed in the brain but show restricted expression patterns
in different subsets of neurons. Moreover, VILIP-1 and -3 show differences in calcium affinities,
membrane binding kinetics and in their intracellular targets to which they associate after calcium
binding. Although both proteins use a similar calcium-myristoyl switch mechanism, the proteins show
different calcium-dependent localization to subcellular compartments when expressed in the same
neuron. These distinct myristoyl switch properties enable the VILIPs to take part in different
signalling cascades in a calcium-dependent manner. For VILIP-1, an involvement in cyclic nucleotide
signalling has been demonstrated, whereas VILIP-3 most likely modulates MAPK activity. Thus, the
highly homologous proteins VILIP-1 and -3 seem to have evolved to fulfil specialised functions in
the neuronal signaling of subsets of neurons in the nervous system. | | | Publisher |
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Braunewell, K.-H., Brackmann, M., Hofmann, A. (2006)
Calcium-binding Proteins 1, 12-15
Abstract
VILIP-1 belongs to the subfamily of Visinin-like proteins with its members VILIP-1, -2, -3, hippocalcin
and neurocalcin delta which have been implicated in the modulation of a variety of intracellular signaling
cascades. VILIP-1 has recently been characterised in more detail and an involvement in cyclic nucleotide
signaling has been demonstrated. Thus, similar to other NCS proteins such as GCAPs and neurocalcin delta,
VILIP-1 can influence the activtiy of guanylyl cyclase enzymes and the cGMP-signaling system of neurons
in the nervous system. | | | Publisher |
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Yusof, A.M., Hu, N.-J., Wlodawer, A., Hofmann, A. (2005)
Proteins 58, 255-262
Abstract
Cyclase-associated protein (CAP) is a highly conserved and widely distributed protein that
links the nutritional response signaling to cytoskeleton remodeling. In yeast, CAP is a
component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic
cycle of the cyclase. While the N-terminal domain of CAP (N-CAP) provides a binding site for
adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity.
Our attempts to crystallise full-length recombinant CAP from Dictyostelium discoideum resulted
in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227),
due to auto-proteolytic cleavage. The structure was solved by molecular replacement with data
at 2.2 Å resolution. The present crystal structure allows the characterisation of a head-to-tail
N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer. Comparison with
previously published structures of N-CAP reveals variable modes of dimerisation of this domain,
but the presence of a common interface for the side-to-side dimer. | | | PubMed | DOI | Publisher |
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Dabitz, N., Hu, N.-J., Yusof, A., Tranter, N., Winter, A., Daley, M., Zschörnig, O., Brisson, A., Hofmann, A. (2005)
Biochemistry 44, 16292-16300
Abstract
The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1
from Gossypium hirsutum, with phospholipid membranes have been characterised using liposome-based
assays and adsorption to monolayers. These two plant annexins show a preference for
phosphatidylserine-containing membranes and display a membrane binding behaviour with a
half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant
annexins also display calcium-independent membrane binding at levels of 10%-20% at neutral pH.
This binding is regulated by three conserved surface-exposed residues on the convex side of the
proteins that play a pivotal role for membrane binding. Due to quantitative differences in the
membrane binding behaviour of N-terminal His-tagged and wild type annexin 24(Ca32), we conclude
that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings
in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane
aggregation and fusion, as well as the effect of these proteins on membrane surface
hydrophobicity, agree with findings from the membrane binding experiments.
Results from electron microscopy reveal elongated rod-like assemblies of plant annexins in the
membrane-bound state. It is possible that these structures consist of protein molecules directly
interacting with the membrane surface and molecules that are membrane-associated but not in direct
contact with the phospholipids. The rod-like structures would also agree with the complex data
from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane
cytoskeleton scaffolding or exocytotic processes.
Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin
fold where these two plant annexins possess distinct modalities compared to mammalian and other
non-plant annexins. | | | PubMed | DOI | PDF |
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Hu, N.-J., Currid, M., Daley, M., Hofmann, A. (2005)
Appl. Spectrosc. 59, 68A
Abstract
Updates of two programmes concerned with automated CD (ACDP) and fluorescence (AFDP) data processing
have been released. | | | Publisher |
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