Biomedical subjects

B Brodsky

Publications and source records attributed to B Brodsky.

At least 37 records · Page 2Linked to original sources

PubMed1998

X-ray crystallographic determination of a collagen-like peptide with the repeating sequence (Pro-Pro-Gly).

The crystal structure of the triple-helical peptide (Pro-Pro-Gly)10 has been re-determined to obtain a more accurate description for this widely studied collagen model and to provide a comparison with the recent high-resolution crystal structure of a collagen-like peptide containing Pro-Hyp-Gly regions. This structure demonstrated that hydroxyproline participates extensively in a repetitive hydrogen-bonded assembly between the peptide and the solvent molecules. Two separate structural studies of the peptide (Pro-Pro-Gly)10 were performed with different crystallization conditions, data collection temperatures, and X-ray sources. The polymer-like structure of one triple-helical repeat of Pro-Pro-Gly has been determined to 2.0 A resolution in one case and 1.7 A resolution in the other. The solvent structures of the two peptides were independently determined specifically for validation purposes. The two structures display a reverse chain trace compared with the original structure determination. In comparison with the Hyp-containing peptide, the two Pro-Pro-Gly structures demonstrate very similar molecular conformation and analogous hydration patterns involving carbonyl groups, but have different crystal packing. This difference in crystal packing indicates that the involvement of hydroxyproline in an extended hydration network is critical for the lateral assembly and supermolecular structure of collagen.

Collagen↗

PubMed1998

Supercoiled protein motifs: the collagen triple-helix and the alpha-helical coiled coil.

The collagen triple-helix and the alpha-helical coiled coil represent the two basic supercoiled multistranded protein motifs. Originally they were characterized in fibrous proteins, but have been found more recently in a number of other proteins containing rod-shaped domains. Coiled-coil domains are responsible for the oligomerization of proteins, as well as other specific functions, while the triple-helix domains associate to form supramolecular structures and bind a variety of ligands. Both structures were originally solved by fiber diffraction, and recent crystallographic studies on small proteins and peptide models have confirmed the structure and provided molecular details. The differences in the molecular conformations of these two motifs and the interactions stabilizing these conformations are discussed. The molecular structures of both motifs constrain the amino acid sequence to recognizable patterns, requiring the (Gly-X-Y)n repeating sequence for the collagen triple-helix and a less stringent heptad repeat requirement (h-x-x-h-x-x-x)n for the coiled-coil domains, where h represents hydrophobic residues. The features and roles of these supercoiled domains in proteins are considered when they are found adjacent in the same protein.

Animals↗

PubMed1998

Gly-X-Y tripeptide frequencies in collagen: a context for host-guest triple-helical peptides.

The collagen triple-helix consists of a repeating (Gly-X-Y)n sequence. In theory, there are more than 400 possible Gly-X-Y triplets, but analysis of sequences from fibrillar and nonfibrillar collagens shows that only a limited set of triplets are found in significant numbers, and many are never observed. The nonrandom frequency of Gly-X-Y triplets makes it practical to experimentally approach the stability of much of the collagen sequence through the study of a limited set of host-guest peptides. In these peptides, individual Gly-X-Y triplets constitute the guest, while the host consists of Gly-Pro-Hyp tripeptides. A set of host-guest peptides was designed to contain the most common nonpolar and charged triplets found in collagen. All formed stable triple-helices, with their melting temperature depending on the identity of the guest triplet. While including less than 10% of all possible triplets, the data set covers 50-60% of collagen sequences and provides a starting point for establishing a stability scale to predict the relative stability of important collagen regions, such as the matrix metalloproteinase cleavage site or binding sites.

Amino Acid Sequence↗

PubMed1998

Skin surface proteolytic activity. Partial characterization and identification.

Skin surface proteolytic activity in the living animal was determined by a sensitive, non-invasive methodology developed in our laboratory. A non-leaky well was constructed on the shaved back of an anesthetized guinea pig. The well contained the reaction mixture including the substrate 125I-S-carboxymethylated insulin B-chain (ICMI). The proteolytic activity was shown to be time-dependent. The activity was strongly inhibited by pepstatin A, indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Pretreatment of the skin with propylene glycol blocked the proteolytic activity. The present study demonstrates the presence of proteolytic activity located on skin surface using a unique, non-invasive method for in situ proteinase determination in the living animal.

Animals↗

PubMed1997

Positional preferences of ionizable residues in Gly-X-Y triplets of the collagen triple-helix.

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

Amino Acids↗

PubMed1997

Gly-Pro-Arg confers stability similar to Gly-Pro-Hyp in the collagen triple-helix of host-guest peptides.

A set of host-guest peptides of the form Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2 has been designed to evaluate the propensity of different Gly-X-Y triplets for the triple-helix conformation (Shah, N. K., Ramshaw, J. A. M., Kirkpatrick, A., Shah, C., and Brodsky, B. (1996) Biochemistry 35, 10262-10268). All Gly-X-Y guest triplets led to a decrease in melting temperature from the host (Gly-Pro-Hyp)8 peptide except for Gly-Pro-Arg. In this Gly-Pro-Hyp-rich environment, Gly-Pro-Arg was found to be as stabilizing as Gly-Pro-Hyp. Decreased stability of host-guest peptides containing Gly-Pro-Lys, Gly-Pro-homo-Arg, and Gly-Arg-Hyp compared with Gly-Pro-Arg indicated a stabilization that is optimal for Arg and specific to the Y-position. Arg was found to have a similar stabilizing effect when residues other than Pro are in the X-position. Both Arg and Hyp stabilize the triple-helix preferentially in the Y-position in a stereospecific manner and occupy largely Y-positions in collagen. However, contiguous Gly-Pro-Hyp units are highly stable and promote triple-helix folding, whereas incorporation of multiple Gly-Pro-Arg triplets was destabilizing and folded slowly due to charge repulsion. In collagen, Gly-Pro-Arg may contribute maximally to local triple-helix stability while also having the potential for electrostatic interactions in fibril formation and binding.

Amino Acid Sequence↗

PubMed1997

Altered collagen structure in mouse tail tendon lacking the alpha 2(I) chain.

Type I collagen is the most prevalent member of the fibril forming family of collagens in higher vertebrates and its heterotrimeric form is comprised of two alpha 1(I) chains and one alpha2(I) polypeptide chain. The functional importance of having two distinct chain types in type I collagen is largely undefined. The existence of a mouse model with a Cola-2 gene mutation (termed oim) that results in non-functional pro alpha 2(I) chains presents a unique opportunity to explore changes in collagen structure resulting from the complete (oim/oim mice) and partial (oim/+ mice) loss of functional alpha 2(I) chains. Tail tendon is a tissue with a well-defined, hierarchical organization of type I collagen. X-ray diffraction studies on oim/oim versus control tendons indicate that the total absence of alpha 2(I) chains results in a decrease in the order of axial packing and a loss of crystalline lateral packing. This suggests that the non-equivalence of three chains is an important determinant of lateral interactions between adjacent molecules and may be involved in the long-range axial order in type I collagen-containing tissues. Both homotrimeric and heterotrimeric type I collagen molecules are found in heterozygous oim mice and these appear to be present in the same co-polymeric fibrils, preventing crystalline lateral packing. In addition to these changes at a fibrillar level, the absence of the alpha 2(I) chain results in an increased enzymatic susceptibility at one site. The oim/oim mice are observed to have reduced body size and smaller tendon bundles, which may be a consequence of these molecular and fibrillar changes in collagen. Furthermore, it is likely that a similar alteration in the molecular packing of collagen in bone fibrils contributes to the osteopenia and decreased bone strength in mice with the oim mutation that are also characteristic of human osteogenesis imperfecta.

Animals↗

PubMed1997

Amino acid sequence environment modulates the disruption by osteogenesis imperfecta glycine substitutions in collagen-like peptides.

Ostoegenesis imperfecta (OI) or "brittle bone" disease is associated with mutations in the genes for type I collagen chains and produces variable phenotypes, ranging from lethal cases at birth to mild cases with increased bone fractures. The most common OI mutations are single base substitutions leading to replacement of Gly by another residue, breaking the typical (Gly-X-Y)n repeating sequence pattern of the collagen triple-helix. Triple-helical peptides were designed to focus on residues 892-921 of the alpha1 chain of type I collagen, where two OI Gly-->Ser mutations are found in close proximity, a mild mutation at site 901 and a lethal mutation at site 913. Peptides were designed to include amino acid sequences around these mutation sites, and were synthesized with the normal sequence or with the Gly-->Ser mutated sequence. The peptide including the normal sequence residues 892-909 with four Gly-Pro-Hyp triplets at the C-terminus formed a stable triple-helix, and introduction of a Ser residue for Gly at the 901 mutation site led to a 50% loss of triple-helix content and a decrease in thermal stability, with little effect on folding. A peptide including residues 904-921 again formed a stable triple-helix, but the introduction of the Gly-->Ser substitution at site 913 led to a much greater decrease in thermal stability. These studies demonstrate the impact of local sequences flanking the Gly substitution on structural consequences and support the concept of variability and regional effects along the collagen molecule.

Amino Acid Sequence↗

PubMed1997

Gly-Gly-containing triplets of low stability adjacent to a type III collagen epitope.

Collagens, in addition to their structural role in the extracellular matrix, possess a number of functional binding domains. In this study, the binding to collagen of a monoclonal antibody is used as a model to define the molecular features involved in triple-helix interactions with other proteins. Here we report the thermal stability of an overlapping set of triple-helical peptides that includes the epitope recognized by a monoclonal antibody to type III collagen. Although the sequences of these peptides are very closely related, by a translation of a single triplet along the collagen chain, substantial variations in the melting temperatures were observed. These variations in thermal stability could not be readily explained by differences in imino acid content, or in numbers of charged or hydrophobic residues. The results indicate that Gly-Gly-Y triplets, which are adjacent to the epitope, have a strong influence in reducing the thermal stability of triple-helical peptides. Further studies, which were carried out on a set of "host-guest" triple-helical peptides containing different Gly-Gly-Y guest triplets, confirm the destabilizing effect of such tripeptides. The presence of Gly-Gly-Y triplets may play an important role in specific functions of type III collagen by modulating the local triple-helical structure or dynamics.

Amino Acid Sequence↗

PubMed1997

Protective effect of povidone-iodine ointment against skin lesions induced by sulphur and nitrogen mustards and by non-mustard vesicants.

Mustard gas (sulphur mustard, SM) is a powerful vesicant employed as a chemical weapon. The present study demonstrates the effect of povidone iodine (PI) ointment against skin toxicity caused by SM. Gross and histopathological examinations showed that application of PI up to 20 min following exposure to the vesicant resulted in marked skin protection. The shorter the interval between exposure and treatment the better was the protection achieved. PI was also effective against other mustards such as carboxybutyl chloroethyl sulphide (CBCS) and mechlorethamine. The fact that PI protected the skin against agents which cannot be oxidized such as iodoacetic acid, divinylsulphone and cantharidine showed that the antidotal effect of PI was unrelated to oxidation of the nitrogen and sulphur atoms of the mustards. PI ointment is proposed as an efficient protective agent against skin toxicity caused by mustards and other alkylators.

Animals↗

PubMed1997

Noninvasive procedure for in situ determination of skin surface aspartic proteinase activity in animals; implications for human skin.

The present study demonstrated a noninvasive procedure for in situ determination of stratum corneum aspartic proteinase in the living animal. A non-leaky well, containing [125I]S-carboxymethylated insulin B-chain (ICMI) as a substrate, was constructed on the shaved back of anesthetized guinea pigs and rats. The enzymatic activity was determined by measuring the radiolabeled trichloroacetic acid soluble material. We demonstrated pepstatin-sensitive proteinase activity bound to the skin surface indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Aged rats had about six fold lower activity than young animals. The proteinase activity was inhibited by the alkylating agent mechlorethamine and by the cosmetic propylene glycol. A similar procedure was carried out with intact human skin pieces obtained during plastic surgery. The activity was inhibited by antihuman cathepsin D antibodies. Cathepsin D was immunohistochemically localized in the corneal and granular layers of the epidermis. Skin surface aspartic proteinase/cathepsin D activity may serve as a marker for skin aging or for certain skin disorders leading to a new approach to their medical treatments.

Aging↗

PubMed1997

The collagen triple-helix structure.

Recent advances, principally through the study of peptide models, have led to an enhanced understanding of the structure and function of the collagen triple helix. In particular, the first crystal structure has clearly shown the highly ordered hydration network critical for stabilizing both the molecular conformation and the interactions between triple helices. The sequence dependent nature of the conformational features is also under active investigation by NMR and other techniques. The triple-helix motif has now been identified in proteins other than collagens, and it has been established as being important in many specific biological interactions as well as being a structural element. The nature of recognition and the degree of specificity for interactions involving triple helices may differ from globular proteins. Triple-helix binding domains consist of linear sequences along the helix, making them amenable to characterization by simple model peptides. The application of structural techniques to such model peptides can serve to clarify the interactions involved in triple-helix recognition and binding and can help explain the varying impact of different structural alterations found in mutant collagens in diseased states.

Amino Acid Sequence↗

PubMed1997

Real-time NMR investigations of triple-helix folding and collagen folding diseases.

Folding of the collagen triple helix provides an opportunity to look at multichain molecular assembly. This triple helix also offers unique advantages for the study of folding because the process is very slow compared to globular proteins, and the kinetics of folding can be obtained in real time by NMR. Studies on triple-helical peptides illustrate the ability to observe kinetic folding intermediates directly and the ability to propose detailed mechanisms of folding through the use of real-time NMR methods. Defective collagen folding has been implicated in various connective tissue diseases and the capacity of NMR to look at the folding of specific sites provides a tool for obtaining information about altered folding mechanisms. Comparison of folding in peptides that model normal and diseased collagens could shed light on the molecular perturbation and the etiology of disease.

Amino Acid Sequence↗

PubMed1996

Binding studies of a triple-helical peptide model of macrophage scavenger receptor to tetraplex nucleic acids.

The macrophage scavenger receptor (MSR), involved in the uptake of oxidized LDL, binds a variety of polyanionic ligands, and in particular shows selectivity for tetraplex forms of nucleic acids. The ligand binding region has been shown to lie in the triple-helical collagen-like domain of MSR. A model peptide-nucleic acid system is presented here to clarify how the triple-helical motif of MSR recognizes and binds tetraplex nucleic acids. The triple-helical peptide MSR-1, with the sequence (POG)3PKGQKGEKG(POG)4, contains a nine amino acid basic sequence implicated in MSR ligand binding, flanked by Pro-Hyp-Gly triplets to provide stability. The ability of this triple-helical MSR-1 peptide to bind to and perturb the conformation of nucleic acids in tetraplex, duplex, and single-stranded states was assessed by monitoring changes in the nucleic acid circular dichroism spectrum in the 240-300 nm region. Our results show that the triple-helical MSR-1 peptide binds to tetraplex poly(I) in a stoichiometric manner and is capable of reproducing the discrimination exhibited by the native MSR molecule for tetraplex over double-stranded or single-stranded nucleic acid states. The triple-helical reference peptide (POG)10 does not bind to tetraplex poly(I), suggesting that binding requires the highly basic 9-mer sequence from MSR that is included in MSR-1. The MSR-1 peptide did not perturb the CD spectra of a series of other tetraplex nucleic acids, indicating that it does not model the broader specificity that MSR shows under physiological conditions. Models of possible interactions between a triple-helical peptide and a tetraplex polynucleotide are proposed on the basis of the stoichiometry observed for the complex between triple-helical MSR-1 and tetraplex poly(I).

Amino Acid Sequence↗

PubMed1996

A host-guest set of triple-helical peptides: stability of Gly-X-Y triplets containing common nonpolar residues.

Host-guest peptide sets have been useful in evaluating the propensity of different amino acids to adopt an alpha-helical or beta-sheet form, and this concept is applied here to the triple-helical conformation. A set of host-guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-GlyCONH2 was designed to evaluate the contribution of an isolated Gly-X-Y triplet to triple-helix stability in a defined environment. Peptides were synthesized to include guest triplets with the X and Y positions occupied by the most common nonpolar residues found in collagen: Pro (X position) and Hyp (Y position); Ala; Leu, the most frequent hydrophobic residue; and Phe, the only commonly occurring aromatic residue. The guest triplets of the 12 peptides synthesized represent 35% of the sequence found in the alpha 1 chain of type I collagen. All peptides formed stable triple-helical structures, and the peptides showed a range of thermal stabilities (Tm = 21-44 degrees C), depending on the identity of the guest triplet. Thermodynamic calculations indicate these peptides have a range of free energy values (delta delta G = 9 kcal/mol) and suggest that favorable entropy is the dominant factor in increased stability. Replacement of Ala by Leu in the X position did not affect the thermal stability, while an Ala to Leu change in the Y position was destabilizing. These data provide experimental evidence that hydrophobic residues do not stabilize the triple helical conformation. Although Leu and Phe are found almost exclusively in the X position in collagens, peptides with Leu and Phe in the Y position formed stable triple-helices. This supports the hypothesis that the X positional preference of these residues relates to their increased potential for intermolecular hydrophobic interactions rather than their destabilization of the triple-helical molecule. These studies establish the utility of host-guest peptides in defining a scale of triple-helix propensities and in clarifying the interactions stabilizing the triple-helical conformation.

Amino Acid Sequence↗

PubMed1996

In vivo and in vitro toxicity of newly synthesized monofunctional sulfur mustard derivatives.

The toxicity of two new monofunctional sulfur mustard derivatives was tested. The compound (4-carboxybutyl 2-chloroethyl sulfide, CBCS; 10-carboxydecyl 2-chloroethyl sulfide, CDCS) possess the 2-chloroethyl sulfide moiety present in mustard gas. Exposure of guinea pig skin to CBCS resulted in a dose-related ulcerative effect. CDCS exhibited similar pathological effects. Dimethylsulfoxide (DMSO) exacerbated CBCS toxicity. Regeneration and healing were prominent six days after application. Concentration-related effects were found in in vitro systems, using human SH-SY5Y neuroblastoma cells for acute toxicity and Y79 retinoblastoma cells for colony forming assay. CBCS or derivatives may serve as models compounds for investigating the mechanism of action of alkylating agents.

Administration, Topical↗

PubMed1996

Direct NMR measurement of folding kinetics of a trimeric peptide.

Direct NMR measurements of the folding kinetics are performed on a collagen-like triple helical peptide. The triple helical peptide was designed to model a biologically important region of collagen and has the sequence (POG)3ITGARGLAG(POG)4. Triple helical peptides were synthesized with specifically labeled 15N amino acid residues in key positions, and the kinetics of folding of the individual residues were monitored directly by measuring the loss of monomer intensity and the increase in trimer intensity. The residues at the terminal ends and central region could be followed independently and quantitated directly. Residues located at the terminal ends have rates and kinetics of folding that are distinct from residues in the central region of the peptide. This allows the monitoring of different steps in the folding mechanism and the postulation of the existence of a kinetic intermediate. The NMR data are consistent with a mechanism of association/nucleation and propagation. Hereditary connective tissue diseases are associated with mutations that result in abnormal folding of collagen, and the NMR folding experiments on a collagen-like peptide provide a basis for characterizing the molecular defect in folding mutations.

Amino Acid Sequence↗

PubMed1996

Memory performance among women with parental abuse histories: enhanced directed forgetting or directed remembering?

Performance on a directed forgetting task was assessed in 24 individuals with borderline personality disorder and early life parental abuse, 24 borderline individuals with no history of abuse, and 24 healthy nonclinical controls under conditions of explicit and implicit memory. In the explicit memory condition, individuals with abuse histories showed greater differential recall of "to-be-remembered" versus "to-be-forgotten" material compared to the 2 comparison groups. Implicit memory performance was equivalent for all 3 groups. The enhanced selective memory in the abused group was the result of better recall for "remember" and not poorer recall for "forget" information, indicating that abused individuals have an enhanced ability to sustain attention to designated "remember" information. Because most people with childhood abuse recall their abuse, enhanced remembering of designated events (e.g., information not associated with abuse) may be a coping strategy.

Adult↗