Biomedical subjects

Fred J Stevens

Publications and source records attributed to Fred J Stevens.

14 recordsLinked to original sources

PubMed2007

Diversity and diversification of light chains in myeloma: the specter of amyloidogenesis by proxy.

BACKGROUND/AIMS: Primary amyloidosis and the cancer, multiple myeloma, are characterized by the overproduction of free antibody light chains. Approximately 10% of myeloma patients develop amyloidosis; primary amyloidosis may be thought of as the pathological analog of monoclonal gammopathy of undetermined significance. The kidney is a common site of accumulation of amyloid fibrils and is also the target of other light chain pathologies. Understanding the structural origin of these pathologies is complicated by the extreme primary structure heterogeneity of light chains. METHODS: Patterns of light chain germline gene usage in myeloma patients were compared to those found in other immune system disorders: lymphoma, leukemia, systemic lupus erythematosus and rheumatoid arthritis. RESULTS: Significant differences in apparent gene usage are found in the various diseases; several germline gene products have not been documented in myeloma patients to date. CONCLUSION: The plasma cell dyscrasias including myeloma, lymphoma, leukemia, and monoclonal gammopathy of undetermined significance are usually monoclonal diseases; however, the light chains produced are not homogeneous. Thus, the pathological risk for the patient may change during the course of the illness. Mutation rates in light chains observed during clonal diversification parallel mutations occurring in all genes in the malignant cells and could be a clinically useful biomarker.

Amino Acid Sequence↗

PubMed2006

Primary structure-based function characterization of BRCT domain replicates in BRCA1.

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.

Amino Acid Sequence↗

PubMed2006

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli.

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.

Amyloid↗

PubMed2005

Truncated wild-type SOD1 and FALS-linked mutant SOD1 cause neural cell death in the chick embryo spinal cord.

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (FALS), and approximately 25% of FALS cases are caused by mutations in superoxide dismutase-1 (SOD1). Mutant (MT) SOD1 kills motor neurons because of the mutant protein's toxicity; however, the basis for toxicity is unknown. We electroporated wild-type (WT), truncated WT or MTSOD1 expression constructs into the chick embryo spinal cord. MTSOD1 and truncated WTSOD1 (as small as 36 amino acid residues in length) aggregated in the cytoplasm of cells and caused cell death. These results suggest that MTSOD1 and truncated WTSOD1 lead to neural cell death because of misfolding, and that SOD1 peptides, possibly as a result of proteolytic digestion of MTSOD, play a role in FALS pathogenesis. Electroporation of the chick embryo spinal cord is a useful system in which to investigate neurodegenerative diseases because it provides efficient delivery of genes into neural cells in situ within a living organism.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Classifying noisy protein sequence data: a case study of immunoglobulin light chains.

SUMMARY: The classification of protein sequences obtained from patients with various immunoglobulin-related conformational diseases may provide insight into structural correlates of pathogenicity. However, clinical data are very sparse and, in the case of antibody-related proteins, the collected sequences have large variability with only a small subset of variations relevant to the protein pathogenicity (function). On this basis, these sequences represent a model system for development of strategies to recognize the small subset of function-determining variations among the much larger number of primary structure diversifications introduced during evolution. Under such conditions, most protein classification algorithms have limited accuracy. To address this problem, we propose a support vector machine (SVM)-based classifier that combines sequence and 3D structural averaging information. Each amino acid in the sequence is represented by a set of six physicochemical properties: hydrophobicity, hydrophilicity, volume, surface area, bulkiness and refractivity. Each position in the sequence is described by the properties of the amino acid at that position and the properties of its neighbors in 3D space or in the sequence. A structure template is selected to determine neighbors in 3D space and a window size is used to determine the neighbors in the sequence. The test data consist of 209 proteins of human antibody immunoglobulin light chains, each represented by aligned sequences of 120 amino acids. The methodology is applied to the classification of protein sequences collected from patients with and without amyloidosis, and indicates that the proposed modified classifiers are more robust to sequence variability than standard SVM classifiers, improving classification error between 5 and 25% and sensitivity between 9 and 17%. The classification results might also suggest possible mechanisms for the propensity of immunoglobulin light chains to amyloid formation.

Algorithms↗

PubMed2004

Mass Spectrometric Evidence for an Alternate Disulfide Bond in Chloroplast Fructose Bisphosphatase.

Mass mapping analysis based on cyanylation (CN) of the protein and CN-induced cleavage indicates that all three cysteine residues in the insertion into the light-activated pea leaf chloroplast fructose bisphosphatase (E.C. 3.1.3.11) are able to participate in disulfide bond formation. There is a major peak in the mass spectrum of the cleavage products indicating that Cys173 forms a disulfide bond with Cys153, consistent with the structure of the oxidized enzyme in PDB files 1d9q and 1dcu, and a minor peak indicating that Cys173 forms an alternate disulfide bond with Cys178. The Cys173-Cys178 disulfide bond was not apparent in the available crystal structures.

CN-induced cleavage↗

PubMed2004

Amyloid formation: an emulation of matrix protein assembly?

Although more than 20 different proteins are now associated with the amyloidoses, the fibrils share many properties. Despite disparity in primary and tertiary structures of the subunit proteins, assembled fibrils exhibit similar morphology, binding of Congo red, interaction with Thioflavine T, formation of complexes with serum amyloid P component, apolipoprotein E, several glycosaminoglycans, the receptor for advanced glycation endproducts and cross-recognition by some monoclonal antibodies. Thus, it is probable that the mechanism of amyloid generation involves a generic process that can be evoked by most, if not all, proteins under conditions that degrade the native conformation. As suggested by others, the beta-helix or beta-roll conformation may be the unifying element of fibril conformations. Several proteins that have evolved to form physiologically useful amyloid-like fibrils, as well as some proteins associated with pathological amyloidoses, exhibit sequence repeat patterns that may facilitate beta-roll or beta-helix formation. Threading analyses of 2 natural amyloid-forming proteins, curli and human Pmel 17, indicate compatibility of their primary structures with both beta sandwich and beta-helix conformations, suggesting a possible innate conformational pliability. In addition, these results may suggest that the misfolded form of some proteins that are associated with conformational disease may be the native conformation of other proteins to which they are linked by evolution. Finally, since many matrix and structural proteins are known to incorporate numerous tandem repeat sequence elements, we propose that the mechanism of fibril formation is fundamentally related to a general protein assembly process that is integral to the generation of cells and tissues.

Amino Acid Sequence↗

PubMed2004

Hypothetical structure of human serum amyloid A protein.

The proteins known as serum amyloid A (SAA) play major, but relatively uncharacterized, roles in the acute phase response and are important components of the innate immune system of humans and probably all vertebrates. N-terminal fragments of the inducible isoforms, SAA1 and SAA2, are the major constituents of fibrils formed during secondary or reactive amyloidosis. Little is known about the structure of SAA beyond secondary structure analyses and circular dichroism spectroscopic data indicating significant alpha helix conformation. Analysis of the primary structure of human SAA indicates probable homology to the N-terminal domain of hemocyanins of arthropods and suggests that approximately 80% of the molecule may consist of a helical bundle with the remaining portion of the C-terminus potentially disordered. This model of SAA suggests that proposed binding sites for laminin, fibronectin, and calcium are segregated to one face of the molecule and that the heparin/heparan binding site is found in the putatively disordered region of the protein. It is possible that removal of the N-terminal 76 amino acid fragment by proteolytic cleavage found generates an unstable entity that undergoes a helix to beta strand transition analogous to the fibril process of A-beta and prion peptides.

Amino Acid Sequence↗

PubMed2004

Analysis of protein-protein interactions by simulation of small-zone gel filtration chromatography.

Small-zone gel filtration chromatography, combined with analytical-scale columns and fast run times, provides a useful system for the study of protein-protein interactions. A computer simulation (SCIMMS, or Simulated Chromatography of Interactive MacroMolecular Systems) that replicates the small-zone behavior of interacting proteins has been developed. The simulation involves an iterative sequence of transport, equilibration, and diffusion steps. This chapter illustrates the use of the simulation to study the homodimerization of rapidly equilibrating immunoglobulin light chain proteins and for determination of association constants. The simulation can also be used to study heterogeneous interactions, kinetically controlled interactions, and higher-order oligomerization, and it can replicate large-zone and Hummel-Dreyer conditions.

Chromatography, Gel↗

PubMed2003

Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation.

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.

Amyloid↗

PubMed2003

Hepatic amyloidosis resulting from deposition of the apolipoprotein A-I variant Leu75Pro.

Apolipoprotein A-I amyloidosis (AApo A-I) is an inherited systemic disease that results from pathologic deposition in tissues of fibrils composed of Apo A-I-related molecules. This disorder has been linked to mutations occurring within the coding region of the Apo A-I gene and heretofore, nine such variants had been described. Recently, a tenth alteration was found in an Italian population where the substitution of proline for leucine at position 75 (Leu75Pro) was associated with amyloid deposits in the liver. We now report our studies on a patient of different ethnicity who has hepatic amyloidosis and a similar mutation in the amyloidogenic precursor protein, as evidenced from analyses of genomic Apo A-I-encoding DNA. Additionally, fibrils extracted from the liver and characterized chemically were found to be composed almost exclusively of a approximately 96 residue N-terminal Apo A-I fragment that contained the Leu75Pro substitution. RFLP analyses revealed that the patient was heterozygous for this mutation; however, < 10% of the plasma Apo A-I consisted of the aberrant protein while the remainder had the normal (wild-type) sequence. Our findings provide further evidence that the Leu75Pro variant is associated with a predominant hepatic phenotype and can occur in individuals of diverse ethnic backgrounds.

Amino Acid Sequence↗

PubMed2002

Kinetics and energetics of assembly, nucleation, and growth of aggregates and fibrils for an amyloidogenic protein. Insights into transition states from pressure, temperature, and co-solute studies.

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.

Amyloid↗

PubMed2002

Support vector machines with selective kernel scaling for protein classification and identification of key amino acid positions.

MOTIVATION: Data that characterize primary and tertiary structures of proteins are now accumulating at a rapid and accelerating rate and require automated computational tools to extract critical information relating amino acid changes with the spectrum of functionally attributes exhibited by a protein. We propose that immunoglobulin-type beta-domains, which are found in approximate 400 functionally distinct forms in humans alone, provide the immense genetic variation within limited conformational changes that might facilitate the development of new computational tools. As an initial step, we describe here an approach based on Support Vector Machine (SVM) technology to identify amino acid variations that contribute to the functional attribute of pathological self-assembly by some human antibody light chains produced during plasma cell diseases. RESULTS: We demonstrate that SVMs with selective kernel scaling are an effective tool in discriminating between benign and pathologic human immunoglobulin light chains. Initial results compare favorably against manual classification performed by experts and indicate the capability of SVMs to capture the underlying structure of the data. The data set consists of 70 proteins of human antibody kappa1 light chains, each represented by aligned sequences of 120 amino acids. We perform feature selection based on a first-order adaptive scaling algorithm, which confirms the importance of changes in certain amino acid positions and identifies other positions that are key in the characterization of protein function.

Algorithms↗

PubMedDate not supplied

Template-based recognition of protein fold within the midnight and twilight zones of protein sequence similarity.

Most homologous pairs of proteins have no significant sequence similarity to each other and are not identified by direct sequence comparison or profile-based strategies. However, multiple sequence alignments of low similarity homologues typically reveal a limited number of positions that are well conserved despite diversity of function. It may be inferred that conservation at most of these positions is the result of the importance of the contribution of these amino acids to the folding and stability of the protein. As such, these amino acids and their relative positions may define a structural signature. We demonstrate that extraction of this fold template provides the basis for the sequence database to be searched for patterns consistent with the fold, enabling identification of homologs that are not recognized by global sequence analysis. The fold template method was developed to address the need for a tool that could comprehensively search the midnight and twilight zones of protein sequence similarity without reliance on global statistical significance. Manual implementations of the fold template method were performed on three folds--immunoglobulin, c-lectin and TIM barrel. Following proof of concept of the template method, an automated version of the approach was developed. This automated fold template method was used to develop fold templates for 10 of the more populated folds in the SCOP database. The fold template method developed three-dimensional structural motifs or signatures that were able to return a diverse collection of proteins, while maintaining a low false positive rate. Although the results of the manual fold template method were more comprehensive than the automated fold template method, the diversity of the results from the automated fold template method surpassed those of current methods that rely on statistical significance to infer evolutionary relationships among divergent proteins.

Amino Acid Sequence↗