Biomedical subjects

Ronald Frank

Publications and source records attributed to Ronald Frank.

At least 19 recordsLinked to original sources

PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗

PubMed2006

Borna disease virus matrix protein is an integral component of the viral ribonucleoprotein complex that does not interfere with polymerase activity.

We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence.

Animals↗

PubMed2006

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Adenosine Triphosphatases↗

PubMed2006

Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor-1alpha.

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.

Amino Acid Sequence↗

PubMed2006

Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface.

Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues (55)DKSRYGGLG(63) of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis.

Antibodies, Bacterial↗

PubMed2006

Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.

Amino Acid Sequence↗

PubMed2005

Ganglioside GM1-binding peptides as adjuvants of antigens inoculated by the intranasal route.

Forty-five GM1-binding peptides were identified using phage-displayed peptides libraries of random peptides. Most have a motif containing a hydrophobic amino acid followed by a serine (S). Based on a GM1-binding assays, two of these GM1-binding peptides (named 15 and 40) were chosen to investigate its immunostimulatory properties when chemically coupled to antigens. Mice intra-nasally (i.n.) vaccinated with some of these complexes developed a better local and systemic antibody response than mice i.n. vaccinated with the respective uncoupled antigens. The efficiency of the complex GM1-binding peptide-antigen strongly depends on the composition and structure of both of the components of the complex.

Adjuvants, Immunologic↗

PubMed2005

Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the alpha-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS alpha-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.

Amino Acid Sequence↗

PubMed2005

Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells.

Protocadherin 12 protein (PCDH12, VE-cadherin 2) is a cell adhesion molecule that has been isolated from endothelial cells. Here, we have used Northern and Western blots, immunohistology, and flow cytometry to examine the distribution of PCDH12 in mouse tissues. It is an N-glycosylated protein of 150-kDa mass. In the endothelium, PCDH12 immunoreactivity was variable and dependent upon the vascular bed. In both the embryo and embryonic stem cell differentiation system, signals were localized in vasculogenic rather than angiogenic endothelium. In addition, the protein was strongly expressed in a subset of invasive cells of the placenta, which were identified as glycogen-rich trophoblasts. In adult mice, strong PCDH12 signals were observed in mesangial cells of kidney glomeruli whereas expression was not detected in other types of perivascular cells. As opposed to most protocadherins, PCDH12 is not expressed in early embryonic (day 12.5) and adult brains. As a first approach to obtain insight into PCDH12 function, we produced transgenic mice deficient in PCDH12, which were viable and fertile. They did not display any obvious histomorphological defects. We conclude that PCDH12 has a unique expression pattern and that its deficiency does not lead to conspicuous abnormalities. Moreover, PCDH12 is the first specific marker for both glycogen-rich trophoblasts and mesangial cells.

Animals↗

PubMed2005

Identification of immunodominant regions of Brassica juncea glyoxalase I as potential antitumor immunomodulation targets.

Glyoxalase I activity has been shown to be directly related to cancer and its inhibitors have been used as anti-cancer drugs. Immunochemical studies have shown immunochemical relatedness among animal and plant glyoxalase I, but its potential application for biomedical research has not been investigated. In order to understand the conserved immunochemical regions of the protein and to determine probable immunomodulation targets, a cellulose-bound scanning peptide library for Brassica juncea glyoxalase I was made using the spot synthesis method. Immuno-probing of the library, using B. juncea anti-glyoxalase I monospecific polyclonal antibodies, revealed three immunodominant regions, epitope I, II, and III. In the homology model of B. juncea glyoxalase I generated by threading its sequence onto the human glyoxalase I, the high accessible surface area and the hydrophilic nature of the epitopes confirmed their surface localization and hence their accessibility for antigen-antibody interaction. Epitopes I and II were specific to B. juncea glyoxalase I. Localizing the epitopes on available glyoxalase I sequences showed that epitope III containing the active site region was conserved across phyla. Therefore, this could be used as a potential immunomodulation target for cancer therapy. Moreover, as the most immunogenic epitopes were mapped on the surface of the protein, this method could be used to discover potential therapeutic targets. It is a simple and fast approach for such investigations. This study, to our knowledge, is the first in epitope mapping of glyoxalase I and has great biomedical potential.

Amino Acid Sequence↗

PubMed2005

The negative regulator of Borna disease virus polymerase is a non-structural protein.

The X protein of Borna disease virus (BDV) negatively regulates viral polymerase activity. With a BDV mini-replicon system, 30 % inhibition of polymerase activity was observed at an X to phosphoprotein (P) plasmid ratio of 1:6 and 100 % inhibition at a ratio of 1:1. It was therefore hypothesized that (i) the X:P ratio in infected cells is not significantly higher than 1:6 to prevent complete inhibition of polymerase activity and (ii) X is not efficiently incorporated into viral particles, allowing efficient replication early in infection. To test these assumptions, a monoclonal antibody directed against BDV X was generated. Immunofluorescence analysis revealed co-localization of X with the nucleoprotein (N) and P in the nucleus, as well as in the cytoplasm of BDV-infected cells. Quantification of viral protein levels by Western blot analysis, using purified Escherichia coli-derived X, P and N as protein standards, revealed an X:P:N ratio in BDV-infected cells of approximately 1:6:40. However, only traces of X could be detected in purified BDV stock, suggesting that X is excluded from virus particles. These results indicate that X is a non-structural protein. The lack of X in virus particles may facilitate polymerase activity early in infection; however, the presence of X in persistently infected cells may result in partial inhibition of the polymerase and thus contribute to viral persistence.

Animals↗

PubMed2004

Systematic peptide array-based delineation of the differential beta-catenin interaction with Tcf4, E-cadherin, and adenomatous polyposis coli.

Nuclear accumulation of the complex between beta-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on beta-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective beta-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the beta-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site.

Animals↗

PubMed2004

Systematic delineation of a calmodulin peptide interaction.

We present a comprehensive profile of amino acid side-chain constraints in a calmodulin (CaM) peptide complex. These data were obtained from the analysis of calmodulin binding to an array of all single substitution analogues as well as N- and C-terminal truncations of the skMLCK derived M13 peptide ligand. The experimentally derived binding data were evaluated with respect to the known 3D-structure of the CaM/M13 complex. Besides an almost perfect agreement between the measured affinities and the structural data, the unexpected high-affine Asn5Ala variant of the M13(*) peptide described by Montigiani et al. could be verified. In contrast to other reports our data clearly support the postulate of the minor and major hydrophobic anchors of this calcium dependent interaction.

Amino Acid Sequence↗

PubMed2004

Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity.

The activity of NF-kappaB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-kappaB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.

Down-Regulation↗

PubMed2004

Binding of alpha2-macroglobulin to GRAB (Protein G-related alpha2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the DeltaA region.

GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42-->Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.

Amino Acid Motifs↗

PubMed2004

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482-789. Talin 482-655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656-789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482-655 fold. We demonstrate that the talin 482-655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

Amino Acid Sequence↗

PubMed2004

PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1alpha from pVHL-independent degradation.

Hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits, serves as a key regulator of metabolic adaptation to hypoxia. The amount of HIF-1alpha protein is regulated either by attenuating von Hippel-Lindau protein (pVHL)-dependent ubiquitination and subsequent 26 S proteasomal degradation or by enhancing cap-dependent mRNA translation, presumably involving a phosphatidyinositol 3-kinase (PI3K)/Akt-regulated pathway. In addition, it became apparent that Hsp90 protects HIF-1alpha from oxygen-independent degradation. Here we present evidence that PI3K/Akt is required for heat shock proteins to stabilize HIF-1alpha. In pVHL-deficient renal cell carcinoma cells, PI3K inhibition by LY294002 and wortmannin or transfection of either a dominant negative PI3K or a kinase-dead Akt mutant substantially lowered constitutively expressed HIF-1alpha without altering HIF-1alpha mRNA. Inhibitors of mitogen-activated protein kinase kinase (MAPKK) such as PD98059 or the p38 MAPK inhibitor SB203580 showed no interference. Considering that PI3K inhibitors down-regulated heat shock protein 90 (Hsp90) as well as Hsp70 expression and moreover attenuated heat- or hypoxia-induced Hsp70 as well as hypoxia-induced Hsp90 up-regulation we conclude that PI3K inhibition promoted degradation of HIF-1alpha indirectly by reducing steady state concentrations of Hsp90 and/or Hsp70. HIF-1alpha co-immunoprecipitated with Hsp90/Hsp70 and direct binding of Hsp70 to the oxygen-dependent degradation domain (ODD) of HIF-1alpha was proven by a pull-down assay and a peptide array. PI3K-mediated degradation of HIF-1alpha was confirmed in HEK 293 cells under hypoxia, suggesting that heat shock proteins constitute an integral component for HIF-1alpha accumulation. We conclude that PI3K/Akt contributes to HIF-1alpha stabilization by provoking expression of heat shock proteins.

Amino Acid Sequence↗

PubMed2004

Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling.

Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF). By screening the other members of SOCS family, we identified SOCS6 as a KIT-binding protein. Using KIT mutants and peptides, we demonstrated that SOCS6 bound directly to KIT tyrosine 567 in the juxtamembrane domain. To investigate the function of this interaction, we constitutively expressed SOCS6 in cell lines. Ectopic expression of SOCS6 in Ba/F3-KIT cell line decreased cell proliferation in response to SCF but not SCF-induced chemotaxis. SOCS6 reduced SCF-induced activation of ERK1/2 and p38 but not activation of AKT or STATs in Ba/F3, murine embryonic fibroblast (MEF), or COS-7 cells. SOCS6 did not impair ERK and p38 activation by other stimuli. These results indicate that SOCS6 binds to KIT juxtamembrane region, which affects upstream signaling components leading to MAPK activation. Our results indicate that KIT signaling is regulated by several SOCS proteins and suggest a putative function for SOCS6 as a negative regulator of receptor tyrosine kinases.

Amino Acid Sequence↗