Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.     

Amino-terminal identity of co-existent amyloid and non-amyloid immunoglobulin kappa light chain deposits. A human disease to study alterations of protein conformation

The sizes of the motor-evoked potentials (MEPs) and the durations of the silent periods after transcranial magnetic stimulation were examined in biceps brachii, brachioradialis and adductor pollicis in human subjects. Stimuli of a wide range of intensities were given during voluntary contractions producing 0-75% of maximal force (maximal voluntary contraction, MVC). In adductor pollicis, MEPs increased in size with stimulus intensity and with weak voluntary contractions (5% MVC), but did not grow larger with stronger contractions. In the elbow flexors, MEPs grew little with stimulus intensity, but increased in size with contractions of up to 50% of maximal. In contrast, the duration of the silent period showed similar changes in the three muscles. In each muscle it increased with stimulus intensity but was unaffected by changes in contraction strength. Comparison of the responses evoked in biceps brachii by focal stimulation over the contralateral motor cortex with those evoked by stimulation with a round magnetic coil over the vertex suggests an excitatory contribution from the ipsilateral cortex during strong voluntary contractions.     

A subset of protein kinase C phosphorylation sites on the myosin II regulatory light chain inhibits phosphorylation by myosin light chain kinase

Protein kinase C (PKC) phosphorylates the regulatory light chains of smooth muscle and cytoplasmic myosin II at three known sites: S1, S2, and T9 [Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1987) J. Biol. Chem. 262, 9569-9573]. Phosphorylation at these sites inhibits the actomyosin ATPase and inhibits phosphorylation of S19 on the regulatory light chain by myosin light chain kinase (MLCK) [Nishikawa, M., Sellers, J. R., Adelstein, R. S., & Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814]. To compare the effects of phosphorylation at a subset of PKC sites on the rate of MLCK phosphorylation, we substituted alanines for the known PKC phosphorylation sites in the Xenopus regulatory light chain (XRLC). PKC phosphorylation of S1A/S2A/T9A revealed secondary phosphorylation sites at T7 and T10, which are accessible both on isolated S1A/S2A/T9A and S1A/S2A/T9A-myosin hybrids. Apparent kinetic constants were determined for MLCK phosphorylation of WT XRLC and XRLC mutants: T9A, S1A/S2A, S1A/S2A/T9A, and T7A/T9A/T10A. PKC prephosphorylation of S1/2 had no effect on the rate of MLCK phosphorylation, while PKC prephosphorylation of T7/9/10 inhibited MLCK phosphorylation due to a 6-fold increase in Km. Our results suggest that phosphorylation of RLC S1/2 as observed in vivo may not be responsible for an inhibition of MLCK phosphorylation.     

The treatment of nephrotic syndrome caused by primary (light chain) amyloid with vincristine, doxorubicin and dexamethasone

Three out of four patients with primary (light chain) amyloid nephrotic syndrome treated with vincristine, doxorubicin and dexamethasone (VAD) induction obtained a partial response and are alive in continuing remission at 4.1, 6.5 and 9.3 years. These preliminary results are of considerable interest and suggest that prospective evaluation of this regimen is warranted in patients with this condition.     

Requirement for Rho-mediated myosin light chain phosphorylation in thrombin-stimulated cell rounding and its dissociation from mitogenesis

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+ mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.     

Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.     

A study on glycosylated high density lipoprotein and its binding to receptors in human lung fibroblasts in diabetic patients

In order to know the glycosylation of high density lipoprotein (HDL) and its specific binding to human lung fibroblasts (HLF) receptors in diabetic patients, the glycosyflation of HDL in diabetic group I (HbAlc < 7.6%) and II (HNbAlc > 9.6%) and a control group were measured with fluorimetry. The hydrazides in carbonyl radical of HDL in the three groups were conjugated with horadish peroxidase (HRP). The specific binding of HDL-HRP to HLF receptors was measured with enzyme linked immunoreceptor assay. The results showed that the glycosylated amount of HDl in group I, II and the control group was 39.26 +/- 8.10, 72.96 +/- 6.40 and 20.40 +/- 1.10 glycogroups/HDL respectively. The surface specific binding of HDL-HRP to HLF receptors in medium with high cholesterol was significantly greater than that in medium without cholesterol (P < 0.01). The surface specific binding of HDL-HRP to HLF receptors in group I and II was significantly lower than that in the control group (P < 0.01) and that in group II was significantly lower than in group I (P < 0.01). The results indicate glycosylated HDL in diabetic patients is increased but its specific binding to HLF receptors is decreased as compared with that in control subjects.     

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.     

Serum amyloid A protein in humans and four animal species: a comparison by two dimensional electrophoresis

Two-dimensional electrophoresis and N-terminal analysis were used to study serum amyloid A protein (SAA) from humans, mink, fox, goat and rabbit. Previously uncharacterized SAA variants were demonstrated in fox, goat and rabbit, and considerable interspecies homology was seen. In rabbit, two novel SAAs were characterized, and SAA1 and SAA2 were demonstrated in mink and rabbit sera. The results confirm previous cDNA studies and indicate that SAA do possess an important function also in fox and goat.     

The effects of excitotoxicity on the expression of the amyloid precursor protein gene in the brain and its modulation by neuroprotective agents

This work has explored the relationship between excitotoxicity and the amyloid precursor protein gene (APP) which may be relevant to future therapeutic developments in Alzheimer's disease. The excitotoxic effects of kainic acid (KA) and pentylenetetrazole (PTZ) have been compared and contrasted on the two major mRNA isoforms of APP using in situ hybridization and quantitative analysis of gene expression in rat brain. The Kunitz Protease Inhibitor containing isoform APP 770 KPI+, the major glial cell isoform, has been shown to be stimulated after KA and was related to neuronal loss and astrocyte activation as gauged by GFAP mRNA. This was associated with reduced expression of APP695 KPI- isoform, the major neuronal isoform. These changes were not observed after PTZ where there was no neuronal loss and no glial reaction. The KA induced changes in APP were prevented by pretreatment with the non-competitive NMDA receptor antagonist dizocilpine and the barbiturate pentobarbitone, but not with the kappa-opioid receptor agonist enadoline. These findings were related to the suppression of seizures and the survival of neurons. In conclusion, excitotoxic stimulation leading to neuronal death was associated with increased expression of APP KPI+ mRNA and decreased APP KPI- mRNA, a finding which may relate to the plasticity of the central nervous system.     

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.     

Characterization of modified staphylococcal protein A containing phospholipid monolayer on both solution and slide surfaces

A method is described for incorporation of water-soluble protein Staphylococcal protein A (SpA) into phospholipid monolayer using covalent protein-lipid conjugates in detergent solution. The amphiphilic conjugates have solubility properties very similar to intergral membrane proteins. When the conjugates are applied into dipalmitoyl-phosphatidic acid monolayer, a protein containing monolayer is formed on subphase surface. The monolayer is transferred to pre-coated substrate surface to form an artificial membrane. Results show that unmodified SpA is readily ejected from the monolayer when compressing the monolayer but modified SpA incorporates into the monolayer stably. The incorporation of the protein is proportional to the lipid coupling degree. When the protein is excessively modified, the IgG binding activity of the SpA in the membrane is lost significantly.     

The acute phase serum amyloid A protein (SAA) in the horse: isolation and characterization of three isoforms

Serum amyloid A (SAA) from acute phase horse serum was isolated using hydrophobic interaction chromatography, gel filtration and ion exchange chromatography. Three SAA isoforms with different isoelectric points, i.e. SAA pI 8.0, SAA pI 9.0 and SAA pI 9.7, were identified by two-dimensional electrophoresis and further characterized with amino acid sequence analysis. These isoforms were found in similar concentrations in all animals investigated, with SAA pI 9.7 constituting about half of the total SAA content. Partial amino acid sequence analysis verified the previously published heterogeneous SAA sequence. SAA pI 8.0 was found to have isoleucine in Position 16, glutamine in Position 44 and glycine in Position 59. SAA pI 9.0 had leucine, glutamine and alanine in the corresponding positions. In SAA pI 9.7 leucine, lysine and alanine were detected. The three isoforms characterized in this study are all acute phase SAAs. SAA pI 9.0 and 9.7 correspond to amyloid A protein variants previously isolated from amyloid deposits of equine liver, while there are no reports on an amyloid A variant corresponding to SAA pI 8.0.     

Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state

The crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE), both with a transition state analog (MgADP x AIF4-) in the active site, have been determined to 2.9 A and 3.5 A resolution, respectively. The MDE structure with an ATP analog (MgADP x BeFx) was also determined to 3.6 A resolution. In all three structures, a domain of the C-terminal region, the "converter," is rotated approximately 70 degrees from that in nucleotide-free skeletal subfragment 1 (S1). We have found that the MDE-BeFx and MDE-AIF4- structures are almost identical, consistent with the fact that they both bind weakly to actin. A comparison of the lever arm positions in MDE-AIF4- and in nucleotide-free skeletal S1 shows that a potential displacement of approximately 10 nm can be achieved during the power stroke.     

The ATPase activity of Myr3, a rat myosin I, is allosterically inhibited by its own tail domain and by Ca2+ binding to its light chain calmodulin

We purified Myr3 (third unconventional myosin from rat), a mammalian "amoeboid" subclass myosin I, from rat liver. The heavy chain of purified Myr3 is associated with a single calmodulin light chain. Myr3 exhibits K/EDTA-ATPase and Mg-ATPase activity. The Mg-ATPase activity is stimulated by increasing F-actin concentrations in a complex triphasic manner similar to the Mg-ATPase activity of myosin I molecules from protozoa. Although purified Myr3 was observed to cross-link actin filaments, it bound in an ATP regulated manner to F-actin, and no evidence for a nucleotide-independent high affinity actin binding site that could explain the triphasic activation pattern was obtained. Micromolar concentrations of free Ca2+ reversibly inhibit the Mg-ATPase activity of Myr3 by binding to its light chain calmodulin, which remains bound to the Myr3 heavy chain irrespective of the free Ca2+ concentration. Polyclonal antibodies and Fab fragments directed against the tail domain were found to stimulate the Mg-ATPase activity. A similar stimulation of the Myr3 Mg-ATPase activity is observed upon proteolytic removal of the very C-terminal SH3 domain. These results demonstrate that Myr3 is subject to negative regulation by free calcium and its own tail domain and possibly positive regulation by a tail-domain binding partner.     

Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.