Biomedical subjects

F J Stevens

Publications and source records attributed to F J Stevens.

At least 19 recordsLinked to original sources

PubMed2002

Increasing protein stability by polar surface residues: domain-wide consequences of interactions within a loop.

We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.

Amino Acid Sequence↗

PubMed2002

Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions.

Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.

Aspartic Acid↗

PubMed2001

Both the environment and somatic mutations govern the aggregation pathway of pathogenic immunoglobulin light chain.

Deposition of monoclonal immunoglobulin light chain (LC) aggregates in tissues is the hallmark of a class of fatal diseases with no effective treatment. In the most prevalent diseases two different types of LC aggregates are observed: fibrillar deposits in LC amyloidosis (AL) and granular aggregates in LC deposition disease (LCDD). The mechanisms by which a given LC forms either type of aggregate are not understood. Although some LCs are more aggregation-prone than others, this does not appear to be due to specific sequence determinants, but more likely from global properties that can be introduced by multiple somatic mutations. Moreover, a single LC isotype can sometimes form both fibrillar and granular aggregates within the same patient. To better understand how the different aggregation pathways arise, we developed a series of in vitro assays to analyze the formation of distinct aggregate types. The recombinant kappa IV LC (SMA) assembles into fibrils when agitated. We now show that SMA can also form granular aggregates upon exposure to copper, and that this aggregation can occur not only in vitro, but also in cells. A constellation of somatic mutations, consisting of His89/His94/Gln96, is sufficient to confer sensitivity to copper on wild-type kappa IV proteins. The formation of both types of aggregates is inhibited by synthetic peptides derived from the LC variable domain. However, the peptide that inhibits fibrillar aggregation is different from the peptide that inhibits copper-induced aggregation. Thus, distinct molecular surfaces of the LC underly each type of aggregate. We conclude that both the intrinsic properties of the sequence and extrinsic conditions govern the aggregation pathway of a LC.

Amino Acid Sequence↗

PubMed2001

Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

Amino Acid Sequence↗

PubMed2001

Counteracting effects of renal solutes on amyloid fibril formation by immunoglobulin light chains.

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.

Amyloid↗

PubMed2001

Caveat receptor: proteomes on display.

Display methods development is currently extending the application of this strategy beyond the generation of ligand binding reagents for research, clinical, or biotechnological purposes to its use as a primary research tool. Peptide- and cDNA display methods have the potential to contribute to understanding the mechanisms of certain classes of drugs and to help map protein-protein interactions of physiological importance. Although the critical contribution of comprehensive amino acid sequence databases has been recognized, of equal importance might be structural genomics concepts in the application of display system technology to proteomics research. Lessons learned from the study of antibody-antigen interactions are reviewed here and applied to the field of display technology with the goal of delineating major factors involved in the successful mimicry of natural protein-ligand interactions.

DNA↗

PubMed2001

Amyloid fibril formation in microwell plates for screening of inhibitors.

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimer's disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis. We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, flavonoids and biflavonoids were screened against fibril formation by a recombinant antibody variable domain (V1). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 microM in 79 microM of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 microM using ThT as a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the V(L) protein dimer. These studies suggested that these compounds could potentially stabilize the VL dimer and therefore reduce its tendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

Amyloid beta-Peptides↗

PubMed2000

Protein conformation and disease: pathological consequences of analogous mutations in homologous proteins.

The antibody light chain variable domain (V(L))(1) and myelin protein zero (MPZ) are representatives of the functionally diverse immunoglobulin superfamily. The V(L) is a subunit of the antigen-binding component of antibodies, while MPZ is the major membrane-linked constituent of the myelin sheaths that coat peripheral nerves. Despite limited amino acid sequence homology, the conformations of the core structures of the two proteins are largely superimposable. Amino acid variations in V(L) account for various conformational disease outcomes, including amyloidosis. However, the specific amino acid changes in V(L) that are responsible for disease have been obscured by multiple concurrent primary structure alterations. Recently, certain demyelination disorders have been linked to point mutations and single amino acid polymorphisms in MPZ. We demonstrate here that some pathogenic variations in MPZ correspond to changes suspected of determining amyloidosis in V(L). This unanticipated observation suggests that studies of the biophysical origin of conformational disease in one member of a superfamily of homologous proteins may have implications throughout the superfamily. In some cases, findings may account for overt disease; in other cases, due to the natural repertoire of inherited polymorphisms, variations in a representative protein may predict subclinical impairment of homologous proteins.

Amino Acid Sequence↗

PubMed2000

Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases.

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.

Catalysis↗

PubMed2000

Immunoglobulin light chains, glycosaminoglycans, and amyloid.

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as beta2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the beta peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue to shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.

Amyloid↗

PubMed2000

Change in dimerization mode by removal of a single unsatisfied polar residue located at the interface.

The importance of unsatisfied hydrogen bonding potential on protein-protein interaction was studied. Two alternate modes of dimerization (conventional and flipped form) of an immunoglobulin light chain variable domain (V(L)) were previously identified. In the flipped form, interface residue Gln89 would have an unsatisfied hydrogen bonding potential. Removal of this Gln should render the flipped dimer as the more favorable quaternary form. High resolution crystallographic studies of the Q89A and Q89L mutants show, as we predicted, that these proteins indeed form flipped dimers with very similar interfaces. A small cavity is present in the Q89A mutant that is reflected in the approximately 100 times lower association constant than found for the Q89L mutant. The association constant of Q89A and Q89L proteins (4 x 10(6) M(-1) and >10(8) M(-1)) are 10- and 1,000-fold higher than that of the wild-type protein that forms conventional dimers clearly showing the energetic reasons for the flipped dimer formation.

Crystallography, X-Ray↗

PubMed2000

Four structural risk factors identify most fibril-forming kappa light chains.

Antibody light chains (LCs) comprise the most structurally diverse family of proteins involved in amyloidosis. Many antibody LCs incorporate structural features that impair their stability and solubility, leading to their assembly into fibrils and to their subsequent pathological deposition when produced in excess during multiple myeloma and primary amyloidosis. The particular amino acid variations in antibody LCs that account for fibril formation and amyloidogenesis have not been identified. This study focuses on amyloidogenesis within the kappa1 family of human LCs. Reanalysis of the current database of primary structures of proteins from more than 100 patients who produced kappa1 LCs, 37 of which were amyloidogenic, reveals apparent structural features that may contribute to amyloidosis. These features include loss of conserved residues or the gain of particular residues through mutation at sites involving a repertoire of approximately 20% of the amino acid positions in the light chain variable domain (V(L)). Moreover 80% of all kappa1 amyloidogenic V(L)s are identifiable by the presence of at least one of three single-site substitutions or the acquisition of an N-linked glycosylation site through mutations. These findings suggest that it is feasible to predict fibril propensity by analysis of primary structure.

Alzheimer Disease↗

PubMed1999

Thermodynamic instability of human lambda 6 light chains: correlation with fibrillogenicity.

Certain types of human light chains have the propensity to deposit pathologically as amyloid fibrils as evidenced by the preferential association of monoclonal lambda 6 proteins with AL amyloidosis. However, the molecular features that render such proteins amyloidogenic have not been elucidated. Based upon the demonstrated relationship between the thermodynamic stability of light chains and their propensity to aggregate in vitro, we have initiated studies where the thermodynamic properties and fibrillogenic potential of two recombinant (r) V lambda 6 molecules were compared. The first protein was generated from cDNA cloned from marrow-derived plasma cells from a patient (Wil) who had AL amyloidosis and renal amyloid deposits; the second was from a patient (Jto) with multiple myeloma in whom the lambda 6 protein was deposited not as amyloid but in the form of renal tubular casts. The thermodynamic stabilities of rV lambda 6Wil and -Jto were determined from chaotropic and thermal denaturation studies. Based upon the Delta GH2O, Delta H, Delta G25 degrees C, Tm, and Cm values, the rV lambda 6Wil was less stable than its nonamyloidogenic counterpart, rV lambda 6Jto. Measurement of fibril formation using a novel in vitro fibril forming assay demonstrated that although both rV lambda 6 proteins formed fibrils in vitro, Wil had a shorter lag time and exhibited faster kinetics under physiologic conditions. Comparative amino acid sequence analyses of these two components and other lambda 6 amyloid-associated light chains revealed that the Jto protein had certain primary structural features that we posit contributed to its increased stability and thus rendered this protein nonamyloidogenic. Our studies provide the first evidence that stabilizing interactions within the V L domain can influence the kinetics of light chain fibrillogenicity.

Amino Acid Sequence↗

PubMed1999

Mapping the major interaction between binding protein and Ig light chains to sites within the variable domain.

Newly synthesized Ig chains are known to interact in vivo with the binding protein (BiP), a major peptide-binding chaperone in the endoplasmic reticulum. The predominant interactions between the light chain and BiP are observed early in the folding pathway, when the light chain is either completely reduced, or has only one disulfide bond. In this study, we describe the in vitro reconstitution of BiP binding to the variable domain of light chains (VL). Binding of deliberately unfolded VL was dramatically more avid than that of folded VL, mimicking the interaction in vivo. Furthermore, VL binding was inhibited by addition of ATP, was competed with excess unlabeled VL, and was demonstrated with several different VL proteins. Using this assay, peptides derived from the VL sequence were tested experimentally for their ability to bind BiP. Four peptides from both beta sheets of VL were shown to bind BiP specifically, two with significantly higher affinity. As few as these two peptide sites, one from each beta sheet of VL, are sufficient to explain the association of BiP with the entire light chain. These results suggest how BiP directs the folding of Ig in vivo and how it may be used in shaping the B cell repertoire.

Animals↗

PubMed1999

The in vivo association of BiP with newly synthesized proteins is dependent on the rate and stability of folding and not simply on the presence of sequences that can bind to BiP.

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine lambdaI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as "kinetic traps." Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Adenosine Triphosphatases↗

PubMed1999

Protein folding in the ER.

The endoplasmic reticulum (ER) is a major protein folding compartment for secreted, plasma membrane and organelle proteins. Each of these newly-synthesized polypeptides folds in a deterministic process, affected by the unique conditions that exist in the ER. An understanding of protein folding in the ER is a fundamental biomolecular challenge at two levels. The first level addresses how the amino acid sequence programs that polypeptide to efficiently arrive at a particular fold out of a multitude of alternatives, and how different sequences obtain similar folds. At the second level are the issues introduced by folding not in the cytosol, but in the ER, including the risk of aggregation in a molecularly crowded environment, accommodation of post-translational modifications and the compatibility with subsequent intracellular trafficking. This review discusses both the physicochemical and cell biological constraints of folding, which are the challenges that the ER molecular chaperones help overcome.

Calcium↗

PubMed1999

Small zone, high-speed gel filtration chromatography to detect protein aggregation associated with light chain pathologies.

Small zone gel filtration chromatography can be used for qualitative and quantitative analysis of protein interactions and aggregation phenomena. The technique is fast, accessible to most laboratories, and can be combined with computer simulation to extract quantitative information from experimental data. The programs KRUNCH and SCIMZ will be furnished on written request to the authors.

Bence Jones Protein↗

PubMed1999

Pathogenic light chains and the B-cell repertoire.

Dysfunctional immunoglobulins (Igs) that are prone to aggregation are unavoidably generated by the diverse repertoire of B cells. Here, Fred Stevens and Yair Argon analyse the patterns of mutations that lead to pathological Igs, account for non-random mutations in human Ig sequences and suggest the exertion of selective forces, which contribute to determining and limiting the Ig repertoire.

B-Lymphocytes↗